U.S. patent application number 13/075774 was filed with the patent office on 2011-08-04 for compositions and methods for tissue repair with extracellular matrices.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Karen Christman, Jessica DeQuach, Jennifer Singelyn.
Application Number | 20110189140 13/075774 |
Document ID | / |
Family ID | 42074182 |
Filed Date | 2011-08-04 |
United States Patent
Application |
20110189140 |
Kind Code |
A1 |
Christman; Karen ; et
al. |
August 4, 2011 |
Compositions and Methods for Tissue Repair with Extracellular
Matrices
Abstract
Described herein are compositions comprising decellularized
cardiac extracellular matrix and therapeutic uses thereof. Methods
for treating, repairing or regenerating defective, diseased,
damaged or ischemic cells, tissues or organs in a subject,
preferably a human, using a decellularized cardiac extracellular
matrix of the invention are provided. Methods of preparing
cardiomyocyte culture surfaces and culturing cells with absorbed
decellularized cardiac extracellular matrix are provided.
Inventors: |
Christman; Karen; (San
Diego, CA) ; Singelyn; Jennifer; (Riverdale, NJ)
; DeQuach; Jessica; (La Jolla, CA) |
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
42074182 |
Appl. No.: |
13/075774 |
Filed: |
March 30, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2009/059015 |
Sep 30, 2009 |
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13075774 |
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61101332 |
Sep 30, 2008 |
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Current U.S.
Class: |
424/93.7 ;
435/402; 435/68.1; 514/21.92 |
Current CPC
Class: |
A61L 2400/06 20130101;
A61K 35/34 20130101; A61P 21/00 20180101; A61L 27/3834 20130101;
A61P 9/06 20180101; A61L 2430/20 20130101; A61L 27/3633 20130101;
A61P 9/00 20180101 |
Class at
Publication: |
424/93.7 ;
514/21.92; 435/68.1; 435/402 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61K 38/02 20060101 A61K038/02; A61K 35/34 20060101
A61K035/34; C12P 21/00 20060101 C12P021/00; C12N 5/077 20100101
C12N005/077 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under grant
No. OD004309 awarded by National Institutes of Health (NIH). The
government has certain rights in the invention.
Claims
1. A composition comprising decellularized extracellular matrix
derived from cardiac or skeletal muscle tissue, wherein said
decellularized extracellular matrix retains native tissue specific
proteins and glycosaminoglycans, and wherein said composition is in
a solution form at a temperature between 20.degree. C.-25.degree.
C. and in a gel form at a temperature greater than 25.degree.
C.
2. The composition of claim 1, wherein said composition comprises
naturally or non-naturally occurring factors that recruit cells
into the composition.
3. The composition of claim 1, wherein said composition is
injectable and is formulated to be delivered through a 27G or
smaller needle for tissue repair or regeneration.
4. The composition of claim 1, wherein said composition further
comprises cells selected from the group consisting of pluripotent
stem cells, multipotent stem cells, cardiomyocytes, cardiac
progenitor cells, skeletal myoblasts, or skeletal muscle progenitor
cells.
5. The composition of claim 1, wherein said composition further
comprises an exogenous therapeutic agent or a polymer in a
therapeutically acceptable formulation.
6. The composition of claim 1, wherein said composition further
comprises cells, drugs, proteins, or polysaccharides that can be
delivered inside, attached to the composition before, during, or
after gelation.
7. The composition of claim 1, wherein said composition comprising
the decellularized extracellular matrix from cardiac tissue is
formulated to coat tissue culture plates to culture cardiomyocytes
or other cardiac cell progenitors.
8. The composition of claim 1, wherein the composition comprising
the decellularized extracellular matrix from cardiac tissue is
formulated to be injected or implanted into the infarct wall
following a myocardial infarction for cardiac tissue repair or
regeneration.
9. The composition of claim 1, wherein said composition comprising
the decellularized extracellular matrix from skeletal muscle tissue
is formulated to coat tissue culture plates to culture skeletal
myoblasts or other skeletal muscle progenitor cells.
10. The composition of claim 1, wherein the composition comprising
the decellularized extracellular matrix from skeletal muscle tissue
is formulated to be injected or implanted in a body for skeletal
muscle repair or regeneration.
11. A method of producing a composition comprising decellularized
extracellular matrix from cardiac or skeletal muscle tissue,
comprising: (a) obtaining a cardiac or skeletal muscle tissue
sample having an extracellular matrix component and a
non-extracellular matrix component; (b) processing the cardiac or
skeletal muscle tissue sample with a detergent to remove the
non-extracellular matrix component to obtain decellularized cardiac
extracellular matrix; and (c) sterilizing the decellularized
cardiac or skeletal muscle extracellular matrix.
12. The method of claim 11, further comprising a step of
lyophilizing and grinding up the decellularized cardiac or skeletal
muscle extracellular matrix.
13. The method of claim 11, further comprising a step of
enzymatically treating the decellularized cardiac or skeletal
muscle extracellular matrix.
14. The method of claim 11, further comprising a step of suspending
and neutralizing said decellularized cardiac or skeletal muscle
extracellular matrix in a saline buffered solution.
15. The method of claim 14, wherein said resulting saline buffered
solution comprising said decellularized cardiac or skeletal muscle
extracellular matrix is injectable through a 27G or smaller needle
at 20.degree. C.-25.degree. C., and spontaneously forms in a gel
form at a temperature greater than 25.degree. C.
16. The method of claim 14, wherein said resulting saline buffered
solution comprising said decellularized cardiac or skeletal muscle
extracellular matrix further comprises cells, drugs, proteins, or
polysaccharides that can be delivered inside, attached to the
solution before, during, or after gelation.
17. The method of claim 14, further comprising placing said
resulting saline buffered solution comprising said decellularized
cardiac or skeletal muscle extracellular matrix into tissue culture
plates or wells to form into an adsorbed matrix for culturing
cells.
18. A method of culturing cells on an adsorbed matrix comprising
the steps of: (a) providing a solution comprising decellularized
extracellular matrix derived from cardiac or skeletal muscle tissue
into a tissue culture device; (b) incubating said tissue culture
device to absorb at least some of the decellularized extracellular
matrix onto the device; (c) removing said solution; and (d)
culturing cells on the adsorbed matrix.
19. The method of claim 18, wherein said cells are cardiomyocytes,
cardiac cell progenitors, or other cell types relevant to cardiac
tissue repair.
20. The method of claim 18, wherein said cells are skeletal
myoblasts, skeletal muscle progenitor cells, or other cell types
relevant to skeletal muscle tissue repair.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This patent application is a continuation of PCT Application
No. PCT/US2009/039823, filed Sep. 30, 2009, which claims priority
benefit of U.S. Provisional Application No. 61/101,332 filed Sep.
30, 2008, each of which is incorporated herein by reference in
their entireties.
BACKGROUND
[0003] Various publications, including patents, published
applications, technical articles and scholarly articles are cited
throughout the specification. Each of these cited publications is
incorporated by reference herein, in its entirety.
[0004] Cardiovascular disease is the leading cause of death in the
United States. The most common cause of cardiovascular disease is
myocardial infarction (MI), which occurs when a coronary artery is
occluded. MI results in the death of cardiomyocytes and
extracellular matrix (ECM) degradation, followed by scar tissue
deposition. Eventually heart failure is onset, and the heart
dilates, leading to decreased pumping efficiency. As there are very
few cardiac progenitors in the heart, and these progenitors do not
divide readily and regeneration of the heart tissue does not occur
naturally. Current treatments for heart failure rely heavily on
invasive surgical procedures and do little to repair damaged heart
tissue.
[0005] More recently investigated procedures utilize the injection
of healthy cells into the left ventricle (LV) infarct wall in an
attempt to regenerate the myocardium, although studies have shown
poor injected cell survival. Cells including adult and embryonic
stem cells, induced pluripotent stem cells, and differentiated
cells such as cardiomyocytes have been typically cultured on
surfaces or scaffolds coated with one, or a few extracellular
matrix proteins. Yet, in vivo, these cells exist in a highly
complex extracellular milieu.
[0006] Some naturally derived materials are currently being
investigated for injection into the myocardium including fibrin,
collagen, alginate, matrigel, and gelatin. None of these provide a
significant amount of the native components of the heart
extracellular matrix. For arrhythmia treatment, current
non-ablative forms include injection of fibrin and cells. Existing
matrices for in vitro cell culture for cardiomyocytes, stem cells,
and other cardiac relevant cells include collagen, laminin,
SURECOAT (CELLUTRON, mixture of collagen and laminin), and
gelatin.
[0007] Current efforts to prevent heart failure after myocardial
infarction have focused on cellular transplantation to replace
necrotic cardiomyocytes, prevent negative left ventricular
remodeling, and regenerate heart tissue. However, without the
proper matrix, cardiomyocyte growth in vitro and survival in vivo
have been poor. There is a need for improved compositions for
cardiac repair, arrhythmia treatment, and cardiac cell culture.
Similarly, there is also a need for improved compositions for
skeletal muscle repair, regeneration and cell culturing.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention provides a composition
comprising decellularized extracellular matrix derived from cardiac
tissue. In some instances, the cardiac tissue is myocardial tissue
and in other instances the tissue is pericardial tissue. The
composition can be injectable. The composition can be formulated to
be in liquid form at room temperature, typically 20.degree. C. to
25.degree. C., and in gel form at a temperature greater than room
temperature or greater than 35.degree. C.
[0009] In some instances, said cardiac tissue is selected from the
group consisting of human hearts, primate hearts, porcine hearts,
bovine hearts, or any other mammalian or animal hearts, including
but not limited to, goat heart, mouse heart, rat heart, rabbit
heart, and chicken heart.
[0010] In some instances, the composition is configured to be
injected into the infarct wall following a myocardial infarction.
In some instances, the composition is configured to be delivered to
a tissue through a small gauge needle (e.g., 27 gauge or smaller).
In some instances, said composition is suitable for implantation
into a patient.
[0011] In some instances, the composition comprises naturally
occurring chemotaxis, growth and stimulatory factors that recruit
cells into the composition. In some instances the composition
comprises native glycosaminioglycans. In some instances, the
composition further comprises non-naturally occurring factors that
recruit cells into the composition.
[0012] In some instances, the composition further comprises a
population of exogenous therapeutic cells. The cells can be stem
cells or other precursors of cardiomyocytes or other
cardiac-related cells.
[0013] In some instances, the composition further comprises a
therapeutic agent, and as such is configured as a drug delivery
vehicle. In some instances, the composition is configured as a
non-destructive conduction block to treat, for example,
arrhythmias. In some instances, the composition is configured to
coat surfaces, such as tissue culture plates or scaffolds, to
culture cardiomyocytes or other cell types relevant to cardiac
repair.
[0014] In one aspect, the invention provides a method of producing
a composition comprising decellularized cardiac extracellular
matrix comprising: obtaining a cardiac tissue sample having an
extracellular matrix component and non-extracellular matrix
component; processing the cardiac tissue sample to remove the
non-extracellular matrix component to obtain decellularized cardiac
extracellular matrix, including extracellular proteins and
polysaccharides; and sterilizing the decellularized cardiac
extracellular matrix. In some instances, said method further
comprises the step of lyophilizing and grinding up the
decellularized cardiac extracellular matrix. In some instances,
said method further comprises the step of enzymatically treating,
solubilizing or suspending the decellularized cardiac extracellular
matrix. In some instances, said decellularized cardiac
extracellular matrix is digested with pepsin at a low pH.
[0015] In some instances, said method further comprises the step of
suspending and neutralizing said decellularized cardiac
extracellular matrix in a solution. In some instances, said
solution is a phosphate buffered solution (PBS) or saline solution
which can be injected through a high gauge needle into the
myocardium. In some instances, said composition is formed into a
gel at body temperature. In some instances, said composition
further comprises cells, drugs, proteins or other therapeutic
agents that can be delivered within or attached to the composition
before, during or after gelation.
[0016] In some instances, said solution is placed into tissue
culture plates or wells, incubated at above 35.degree. C. or about
37.degree. C. to form into a gel that is used for cell culture. In
one aspect, the invention provides a method of culturing cells on
an adsorbed matrix comprising the steps of: providing a solution
comprising decellularized extracellular matrix derived from cardiac
tissue into a tissue culture device; incubating said tissue culture
plates device; removing said solution; and culturing cells on the
adsorbed matrix. In some instances, said cells are cardiomyocytes
or other cell types relevant to cardiac repair.
[0017] In one aspect, the invention provides a therapeutic method
for cardiac tissue repair in a subject comprising injecting or
implanting a therapeutically effective amount of a composition
comprising decellularized extracellular matrix derived from cardiac
tissue into a subject in need thereof.
[0018] In another aspect, a composition herein comprises
decellularized extracellular matrix derived from skeletal muscle
tissue. The composition can be injectable. The composition can be
liquid at room temperature and is in a gel form at temperatures
greater than room temperature. In some instances, the composition
is configured to be injected into the infarct wall following a
myocardial infarction. In some instances, the composition is
configured to be delivered to a tissue through a 27g or smaller
needle.
[0019] In some embodiments, the composition comprising
decellularized extracellular matrix derived from skeletal muscle
tissue herein retains native glycosaminoglycans. In some instances,
the composition comprises naturally occurring factors that recruit
cells into the composition. In some instances, the composition
comprises non-naturally occurring factors that recruit cells into
the composition. In some instances, said composition is configured
to coat tissue culture surfaces or scaffolds to culture cells
relevant to skeletal muscle repair.
[0020] In an aspect, a method of producing a composition is
disclosed herein that comprises decellularized skeletal muscle
extracellular matrix comprising: obtaining from a subject a
skeletal muscle tissue sample having an extracellular matrix and
non-extracellular matrix components; processing skeletal muscle
tissue sample to remove the non-extracellular matrix component to
obtain decellularized skeletal muscle extracellular matrix and
extracellular proteins and polysaccharides; and sterilizing the
decellularized skeletal muscle extracellular matrix. In some
instances, said method further comprises the step of lyophilizing
and grinding up the decellularized skeletal muscle extracellular
matrix. In some instances, said method further comprises the step
of enzymatically treating, solubilizing, or suspending the
decellularized skeletal muscle extracellular matrix. In some
instances, said decellularized skeletal muscle extracellular matrix
is digested with pepsin at a low pH. In some instances, said method
further comprises the step of suspending and neutralizing or
altering the pH of said decellularized cardiac extracellular matrix
in a solution. In some instances, said solution is a PBS, saline or
other buffer solution configured to be injected through a small
diameter needle into the myocardium. The solution can be formed
into a gel at body temperature. The solution can further comprise
cells, drugs, proteins, or polysaccharides that can be delivered
inside, attached to the material before, during, or after gelation.
In some instances, the solution is placed into tissue culture
plates or wells, incubated at 37.degree. C., or temperature above
room temperature, to form into a gel that is used for cell
culture.
[0021] In an aspect, a method of culturing cells on an adsorbed
matrix comprises the steps of: providing a solution comprising
decellularized extracellular matrix derived from skeletal muscle
tissue into a tissue culture device; incubating said tissue culture
plates device; removing said solution; and culturing cells on the
adsorbed matrix. In some instances, said cells are skeletal
myoblasts, stem cells or other cell types relevant to skeletal
muscle repair.
[0022] In an aspect, a therapeutic method for skeletal muscle
repair in a subject comprises implanting a composition comprising
decellularized extracellular matrix derived from skeletal muscle
tissue.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 illustrates an exemplary heart resulting from the
method of delivering a composition of the present invention at top
or a standard therapy at bottom.
[0024] FIG. 2 illustrates the average myofibrillar area of human
embryonic stem cell derived cardiomyocytes grown on cardiac
ECM.
[0025] FIG. 3 illustrates the average number of human embryonic
stem cell derived cardiomyocyte nuclei per myofibrillar area grown
on cardiac ECM.
[0026] FIG. 4 shows average desmosome plaque size on cardiac
ECM.
[0027] FIG. 5 illustrates skeletal myoblasts cultured on skeletal
muscle matrix.
[0028] FIG. 6 illustrates that skeletal myoblasts migrate
specifically towards skeletal muscle matrix.
DETAILED DESCRIPTION OF THE INVENTION
[0029] In certain preferred embodiments, the present invention
provides a decellularized cardiac extracellular matrix (ECM)
composition which can be used, for example, to deliver therapeutic
agents, including cells, into the heart wall following a myocardial
infarction. The ECM of the present invention can be derived from
the native or natural matrix of mammalian heart tissue. Described
herein are compositions comprising cardiac ECM which can be used
for injection into cardiac tissue in need of therapeutic treatment.
The ECM can also be used to recruit cells into the injured tissue
or as a drug delivery vehicle. The composition can also be used to
support injured tissue or change the mechanical properties. Another
use of the present invention is as a non-destructive conduction
block to treat, for example, arrhythmias. In some instances, heart
or cardiac ECM as described herein is derived from myocardial
tissue. In other instances, heart or cardiac ECM as described
herein is derived from pericardial tissue.
[0030] A composition comprising the decellularized cardiac ECM as
described herein can help regenerate defective or absent myocardium
and restore cardiac function. The ECM composition can be derived
from an animal or synthetic source. An extracellular matrix
composition herein can further comprise one or more additional
components, for example without limitation: an exogenous cell, a
peptide, polypeptide, or protein, a vector expressing a DNA of a
bioactive molecule, and other therapeutic agents such as drugs,
cellular growth factors, nutrients, antibiotics or other bioactive
molecules. Therefore, in certain preferred embodiments, the ECM
composition can further comprise an exogenous population of cells
such as cardiomyocyte precursors, as described below.
[0031] In some instances, methods of delivery are described wherein
the composition can be placed in contact with a defective, diseased
or absent myocardium, resulting in myocardial tissue regeneration
and restoration of contractility, conductivity, or healthy function
to the heart muscle. In some instances, the composition herein can
recruit endogenous cells within the recipient and can coordinate
the function of the newly recruited or added cells, allowing for
cell proliferation or migration within the composition.
[0032] Prior efforts to prevent heart failure after myocardial
infarction (MI) have focused on cellular transplantation to replace
necrotic cardiomyocytes, prevent negative left ventricular (LV)
remodeling, and regenerate heart tissue. A variety of cell types
have been explored as cellular transplantation therapies, including
cardiomyocytes, skeletal myoblasts, mesenchymal and embryonic stem
cells. Unfortunately, without the proper matrix, cellular survival
in vivo has been poor. Some naturally derived matrices that have
been used to attempt to aid in cell retention and survival upon
injection in the prior art include fibrin, collagen, matrigel,
alginate, and gelatin. However, none of these materials adequately
mimics the native components found specifically in the cardiac
extracellular matrix.
[0033] Current injectable scaffolds to treat the heart post-MI fail
to provide all desired components of the extracellular matrix that
cells require to thrive. Thus, cell survival in such scaffolds has
been limited. In certain embodiments, this invention provides a
native cardiac ECM decellularization and gelation method to create
an in situ scaffold for cellular transplantation. An appropriate
digestion and preparation protocol has been provided herein that
can create nanofibrous gels. The gel solution is capable of being
injected into the myocardium or infarct, thus demonstrating its
potential as an in situ gelling scaffold. Since a decellularized
cardiac ECM best mimics the natural cardiac environment, it
improves cell survival and retention upon injection at the site of
myocardial infarction, thus encouraging myocardial tissue
regeneration.
[0034] FIG. 1 illustrates an exemplary method of delivering a
composition herein. A healthy heart is shown on the left. After
myocardial infarction shown in the central diagram, no current
standard therapies, such as available pharmaceuticals and medical
devices alone, effectively avoid the death of the cardiomyocytes,
negative LV remodeling, LV dilation, and heart failure, as shown in
the bottom right schematic. The present invention ameliorates this
problem by delivering an injectable composition as described
herein. Delivering a composition herein to a LV provides increased
regeneration, reduced infarct size, reduced LV remodeling, and
improved cardiac function, as shown in the upper right schematic
diagram of the heart.
[0035] The invention features decellularized cardiac extracellular
matrix, as well as methods for the production and use thereof. In
particular, the invention relates to a biocompatible composition
comprising decellularized cardiac extracellular matrix derived
directly from cardiac tissue, and is used for treating defective,
diseased, damaged or ischemic tissues or organs in a subject,
preferably a human heart, by injecting or implanting the
biocompatible composition comprising the decellularized cardiac
extracellular matrix into the subject. Other embodiments of the
invention concern decellularized skeletal muscle, extracellular
matrix compositions, methods of use and methods of production
[0036] In some instances, the decellularized cardiac extracellular
matrix is derived from native cardiac tissue selected from the
group consisting of human, porcine, bovine, goat, mouse, rat,
rabbit, chicken or any other mammalian or animal hearts. In some
embodiments, the biocompatible composition comprising the
decellularized cardiac extracellular matrix is in an injectable gel
or solution form, and can be used for cardiac repair by
transplanting or delivering cells contained therein into the
infarct wall following a myocardial infarction, or recruiting the
patient's own cells into the injured cardiac tissue. In other
instances, the biocompatible material comprising a decellularized
cardiac ECM is, for example, a patch, an emulsion, a viscous
liquid, fragments, particles, microbeads, or nanobeads.
[0037] In some instances, the invention provides biocompatible
materials for culturing cardiomyocytes or other cardiac relevant
cells in research laboratories, or in a clinical setting prior to
transplantation and for cardiac repair. Methods for manufacturing
and coating a surface, such as tissue culture plates or wells, with
decellularized cardiac extracellular matrix are also provided. The
biocompatible materials of the invention are also suitable for
implantation into a patient, whether human or animal.
[0038] The invention further provides a method of producing a
biocompatible material comprising the decellularized cardiac
extracellular matrix of the invention. Such method comprises the
steps of: (a) obtaining a cardiac tissue sample having an
extracellular matrix component and non-extracellular matrix
component; (b) processing the cardiac tissue sample to remove at
least a portion or substantially all of the non-extracellular
matrix component to obtain decellularized cardiac extracellular
matrix; and (c) sterilizing the decellularized cardiac
extracellular matrix. In certain embodiments, the cardiac tissue
sample is isolated from a mammal such as a non-primate (e.g., cows,
pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey
and human), or an avian source (e.g., chicken, duck, etc.).
Decellularization procedures for the cardiac tissue sample are
performed using one or more physical, chemical and/or biological
techniques, known in the art and as taught herein.
[0039] For human therapy, there are many potential sources for the
cardiac extracellular matrix material: human heart (including
autologous, allogeneic, or cadaveric), porcine heart, bovine heart,
goat heart, mouse heart, rat heart, rabbit heart, chicken heart,
and other animal sources. Unlike total heart transplantation, one
donor heart can be used to treat many people. Non-human animals are
a source of heart extracellular matrix without the need for human
donors. As a research reagent, non-human animal sources can be
utilized.
[0040] In certain embodiments, the method of processing the cardiac
extracellular matrix is as follows. The heart tissue is first
decellularized, leaving only the extracellular matrix.
Decellularization can be performed with a perfusion of sodium
dodecyl sulfate and phosphate buffered solution, for example. The
heart extracellular matrix is then lyophilized, ground up, and
digested with pepsin at a low pH, between about pH 1-6 or pH 1-4,
or other matrix degrading enzymes such as matrix
metalloproteinases.
[0041] To produce a gel form of the cardiac extracellular matrix
for in vivo therapy, the solution comprising the heart
extracellular matrix is then neutralized and brought up to the
desired temperature, concentration and viscosity using PBS/saline.
In certain embodiments, the ECM concentration can be 1-20 mg/mL, or
2-8 mg/mL. The solution comprising the heart extracellular matrix
can then be injected through a high gauge needle, such as 27 gauge
or higher, into the myocardium. At body temperature, e.g.,
36.8.degree. C..+-.0.7.degree. C., such solution then forms into a
gel. Cells, drugs, proteins, or other therapeutic agents can also
be delivered inside the cardiac ECM gel.
[0042] To produce a gel form of the cardiac extracellular matrix
for in vitro uses, the solution comprising the heart extracellular
matrix is neutralized and brought up to the desired concentration
using PBS/saline. In certain embodiments, the ECM concentration can
be 1-20 mg/mL, or 2-8 mg/mL. Such solution can then be placed onto
any solid surface such as into tissue culture plates/wells. Once
placed in an incubator at 37.degree. C. or above room temperature,
the solution forms a gel that can be used for cell culture.
[0043] The invention also provides a therapeutic method for cardiac
repair in a subject comprising injecting or implanting in part or
in its entirety the biocompatible cardiac ECM material of the
invention into a patient. The invention further provides a
therapeutic method for treating arrhythmia or other defective,
diseased, damaged or ischemic tissue or organ in a subject
comprising injecting or implanting the biocompatible material of
the invention in situ.
[0044] The compositions herein can comprise a decellularized ECM
derived from cardiac tissue and another component or components. In
some instances, the amount of ECM in the total composition is
greater than 90% or 95% or 99% of the composition by weight. In
some embodiments, the ECM in the total composition is greater than
1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the
composition by weight.
[0045] Decellularized extracellular matrices are prepared such that
much of the bioactivity for myocardial tissue regeneration is
preserved. Exemplary bioactivity of the compositions herein include
without limitation: control or initiation of cell adhesion, cell
migration, cell differentiation, cell maturation, cell
organization, cell proliferation, cell death (apoptosis),
stimulation of angiogenesis, proteolytic activity, enzymatic
activity, cell motility, protein and cell modulation, activation of
transcriptional events, provision for translation events,
inhibition of some bioactivities, for example inhibition of
coagulation, stem cell attraction, chemotaxis, and MMP or other
enzyme activity.
[0046] The compositions comprise an extracellular matrix that is
substantially decellularized. In some instances, a decellularized
matrix comprises no living native cells with which the ECM
naturally occurs. In some instances, a substantially decellularized
matrix comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or
10% native cells by weight.
[0047] As described herein, a composition can comprise a
decellularized cardiac ECM and different tissue decellularized EMC
or a synthetic or naturally occurring polymer. Exemplary polymers
herein include, but are not limited to: polyethylene terephthalate
fiber (DACRON), polytetrafluoroethylene (PTFE),
glutaraldehyde-cross linked pericardium, polylactate (PLA),
polyglycol (PGA), hyaluronic acid (HA), polyethylene glycol (PEG),
polyethelene, nitinol, and collagen from animal and non-animal
sources (such as plants or synthetic collagens). In some instances,
a polymer of the composition is biocompatible and biodegradable
and/or bioabsorbable. Exemplary biodegradable or bioabsorbable
polymers include, but are not limited to: polylactides,
poly-glycolides, polycarprolactone, polydioxane and their random
and block copolymers. A biodegradable and/or bioabsorbable polymer
can contain a monomer selected from the group consisting of a
glycolide, lactide, dioxanone, caprolactone, trimethylene
carbonate, ethylene glycol and lysine.
[0048] The polymer material can be a random copolymer, block
copolymer or blend of monomers, homopolymers, copolymers, and/or
heteropolymers that contain these monomers. The biodegradable
and/or bioabsorbable polymers can contain bioabsorbable and
biodegradable linear aliphatic polyesters such as polyglycolide
(PGA) and its random copolymer poly(glycolide-co-lactide-)
(PGA-co-PLA). Other examples of suitable biocompatible polymers are
polyhydroxyalkyl methacrylates including ethylmethacrylate, and
hydrogels such as polyvinylpyrrolidone and polyacrylamides. Other
suitable bioabsorbable materials are biopolymers which include
collagen, gelatin, alginic acid, chitin, chitosan, fibrin,
hyaluronic acid, dextran, polyamino acids, polylysine and
copolymers of these materials. Any combination, copolymer, polymer
or blend thereof of the above examples is contemplated for use
according to the present invention. Such bioabsorbable materials
may be prepared by known methods.
[0049] Therefore, methods are described herein for preparing a
composition comprising decellularized ECM derived from cardiac
muscle tissue. The invention also provides ECM compositions and
methods derived from skeletal muscle tissue in an analogous
process. Related compositions, devices and methods of production
and use also are provided.
[0050] In certain embodiments, the viscosity of the composition
increases when warmed above room temperature including
physiological temperatures approaching about 37.degree. C.
According to one non-limiting embodiment, the ECM-derived
composition is an injectable solution at room temperature and other
temperatures below 35.degree. C. In another non-limiting embodiment
the gel can be injected body temperature above about 37.degree. C.
or near body temperature, but gels more rapidly at increasing
temperatures. A gels forms after approximately 15-20 minutes at
physiological temperature of 37.degree. C. A general set of
principles for preparing an ECM-derived gel is provided along with
preferred specific protocols for preparing gels in the following
Examples which are applicable and adaptable to numerous tissues
including without limitation heart and skeletal muscle.
[0051] The compositions which may include cells or other
therapeutic agents may be implanted into a patient, human or
animal, by a number of methods. In some instances, the compositions
are injected as a liquid into a desired site in the patient.
[0052] Commercially available ECM preparations can also be combined
in the methods, devices and compositions described herein. In one
embodiment, the ECM is derived from small intestinal submucosa
(SIS). Commercially available preparations include, but are not
limited to, SURGISIS.TM., SURGISIS-ES.TM., STRATASIS.TM., and
STRATASIS-ES.TM. (Cook Urological Inc.; Indianapolis, Ind.) and
GRAFTPATCH.TM. (Organogenesis Inc.; Canton, Mass.). In another
embodiment, the ECM is derived from dermis. Commercially available
preparations include, but are not limited to PELVICOL.TM. (sold as
PERMACOL.TM. in Europe; Bard, Covington, Ga.), REPLIFORM.TM.
(Microvasive; Boston, Mass.) and ALLODERIVI.TM. (LifeCell;
Branchburg, N.J.).
[0053] In some instances, the solution, gel form, and adsorbed form
of the heart extracellular matrix of the invention provide all the
constituents at the similar ratios found in vivo. For arrhythmia
treatment, the extracellular matrix of the invention can be
delivered which can allow for cardiac tissue regeneration after
resolution of the arrhythmia. For in vitro cell culture for
cardiomyocytes and other cardiac relevant cells, the gel and
adsorbed forms of the heart extracellular matrix of the invention
contain all or many of the same extracellular matrix cues that the
cells recognize in vivo as compared to the commonly used collagen,
laminin, SURECOAT (CELLUTRON, mixture of collagen and laminin), and
gelatin.
[0054] The compositions herein provide a gel or solution form of
heart extracellular matrix, and the use of these forms of heart
extracellular matrix for cardiac repair, arrhythmia treatment, and
cell culture for example. In one embodiment, the heart tissue is
first decellularized, leaving only the extracellular matrix. The
matrix is then lyophilized, ground or pulverized into a fine
powder, and solubilized with pepsin or other enzymes, such as, but
not limited to, matrix metalloproteases, collagenases, and
trypsin.
[0055] For gel therapy, the solution is then neutralized and
brought up to the appropriate concentration using PBS/saline. In
one embodiment, the solution can then be injected through a needle
into the myocardium (either via cathether, through the ribs, or
during an open chest procedure. The needle size can be without
limitation 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, or smaller.
In one embodiment, the needle size through which the solution is
injected is 27g. Delivery can also occur through a balloon infusion
catheter or other non-needle cathether. Dosage amounts and
frequency can routijnely be determined based on the varying
condition of the injured tissue and patient profile. At body
temperature, the solution can then form into a gel. In yet another
embodiment, gel can be crosslinked with glutaraldehye,
formaldehyde, bis-NHS molecules, or other crosslinkers.
[0056] In yet another embodiment, the ECM can be combined with
other therapeutic agents, such as cells, peptides, proteins, DNA,
drugs, nutrients, antibiotics, survival promoting additives,
proteoglycans, and/or glycosaminolycans. In yet another embodiment,
the ECM can be combined and/or crosslinked with a synthetic
polymer. Examples of synthetic polymers include, but are not
limited to: polyethylene terephthalate fiber (DACRON.TM.),
polytetrafluoroethylene (PTFE), polylactic acid (PLA), polyglycolic
acid (PGA), polyethylene glycol (PEG), polyethylene glycol
diacrylate (PEGDA), polyethylene, polystyrene and nitinol.
[0057] In yet another embodiment, ECM solution or gel can be
injected into the infarct area, border zone, or myocardium alone or
in combination with above-described components for endogenous cell
ingrowth, angiogenesis, and regeneration. In yet another
embodiment, the composition can also be used alone or in
combination with above-described components as a matrix to change
mechanical properties of the heart and/or prevent negative left
ventricular remodeling. In yet another embodiment, the composition
can be delivered with cells alone or in combination with the
above-described components for regenerating myocardium. In yet
another embodiment, the composition can be used alone or in
combination with above-described components for creating a
conduction block to treat arrhythmias.
[0058] In one embodiment for making a soluble reagent, the solution
is brought up in a low pH solution including but not limited to 0.5
M, 0.1, or 0.01 M acetic acid or 0.1M HCl to the desired
concentration and then placed into tissue culture plates/wells,
coverslips, scaffolding or other surfaces for tissue culture. After
placing in an incubator at 37.degree. C. for 1 hour, or overnight
at room temperature, the excess solution is removed. After the
surfaces are rinsed with PBS, cells can be cultured on the adsorbed
matrix. The solution can be combined in advance with peptides,
proteins, DNA, drugs, nutrients, survival promoting additives,
proteoglycans, and/or glycosaminoglycans before, during, or after
injection/implantation.
[0059] The present invention provides enhanced cell attachment and
survival on both the therapeutic composition and adsorbed cell
culturing composition forms of the heart extracellular matrix in
vitro. The soluble cell culturing reagent form of the heart
extracellular matrix induces faster spreading, faster maturation,
and/or improved survival for cardiomyocytes compared to standard
plate coatings.
[0060] Previous studies have shown that is difficult to use human
embryonic stem cell (hESC) derived cardiomyocytes for treatment of
myocardial infarction. In some instances, efficient differentiation
and in vivo yield of mature ventricular cardiomyocytes has hampered
the effectiveness of treatment. Previously, modulation of
differentiation has been largely addressed in vitro, for example,
with addition of soluble factors to cell culture media. This
process has been limited by difficulty in differentiating beyond a
fetal phenotype.
[0061] In addition to soluble factors, extracellular matrix can
also play a large role in cell differentiation. Some matrices
comprising chemical cues have been investigated for adult cells,
including adult progenitors, however limited work has been
performed on ECM effects on ESCs, particularly for hESCs. In many
instances, hESC derived cardiomyocytes are delivered in a
pro-survival mixture consisting of soluble factors and
matrigel.
[0062] In an embodiment herein, a biomimetic matrix derived from
native cardiac tissue is disclosed. In some instances, a matrix
resembles the in vivo cardiac environment in that it contains many
or all of the native chemical cues found in natural cardiac ECM. In
some instances, through crosslinking or addition or other
materials, the mechanical properties of healthy adult or embryonic
myocardium can also be mimicked. As described herein, cardiac ECM
can be isolated and processed into a gel using a simple and
economical process, which is amenable to scale-up for clinical
translation.
[0063] In some instances, a composition as provided herein can
comprise a matrix and exogenously added or recruited cells. The
cells can be any variety of cells. In some instances, the cells are
a variety of cardiac or cardiovascular cells including, but not
limited to: stem cells, progenitors, cardiomyocytes, vascular
cells, and fibroblasts derived from autologous or allogeneic
sources.
[0064] The invention thus provides a use of a gel made from native
decellularized cardiac extracellular matrix to support isolated
neonatal cardiomyocytes or stem cell progenitor derived
cardiomyocytes in vitro and act as an in situ gelling scaffold,
providing a natural matrix to improve cell retention and survival
in the left ventricle wall. A scaffold created from cardiac ECM is
well-suited for cell transplantation in the myocardium, since it
more closely approximates the in vivo environment compared to
currently available materials.
[0065] A composition herein comprising cardiac ECM and exogenously
added cells can be prepared by culturing the cells in the ECM. In
addition, where proteins such as growth factors are added into the
extracellular matrix, the proteins may be added into the
composition, or the protein molecules may be covalently or
non-covalently linked to a molecule in the matrix. The covalent
linking of protein to matrix molecules can be accomplished by
standard covalent protein linking procedures known in the art. The
protein may be covalently or linked to one or more matrix
molecules.
[0066] In one embodiment, when delivering a composition that
comprises the decellularized cardiac ECM and exogenous cells, the
cells can be from cell sources for treating the myocardium that
include allogenic, xenogenic, or autogenic sources. Accordingly,
embryonic stem cells, fetal or adult derived stem cells, induced
pluripotent stem cells, cardio-myocyte progenitors, fetal and
neonatal cardiomyocytes, myofibroblasts, myoblasts, mesenchymal
cells, parenchymal cells, epithelial cells, endothelial cells,
mesothelial cells, fibroblasts, hematopoetic stem cells, bone
marrow-derived progenitor cells, skeletal cells, macrophages,
adipocytes, and autotransplanted expanded cardiomyocytes can be
delivered by a composition herein. In some instances, cells herein
can be cultured ex vivo and in the culture dish environment
differentiate either directly to heart muscle cells, or to bone
marrow cells that can become heart muscle cells. The cultured cells
are then transplanted into the mammal, either with the composition
or in contact with the scaffold and other components.
[0067] Adult stem cells are yet another species of cell that can be
part of a composition herein. Adult stem cells are thought to work
by generating other stem cells (for example those appropriate to
myocardium) in a new site, or they differentiate directly to a
cardiomyocyte in vivo. They may also differentiate into other
lineages after introduction to organs, such as the heart. The adult
mammal provides sources for adult stem cells in circulating
endothelial precursor cells, bone marrow-derived cells, adipose
tissue, or cells from a specific organ. It is known that
mononuclear cells isolated from bone marrow aspirate differentiate
into endothelial cells in vitro and are detected in newly formed
blood vessels after intramuscular injection. Thus, use of cells
from bone marrow aspirate can yield endothelial cells in vivo as a
component of the composition. Other cells which can he employed
with the invention are the mesenchymal stem cells administered with
activating cytokines. Subpopulations of mesenchymal cells have been
shown to differentiate toward myogenic cell lines when exposed to
cytokines in vitro.
[0068] Human embryonic stem cell derived cardiomyocytes can be
grown on a composition herein comprising a cardiac matrix. In some
instances, hESC-derived cardiomyocytes grown in the presence of a
composition herein provide a more in vivo-like morphology. In some
instances, hESC-derived cardiomyocytes grown in the presence of a
composition herein provide increased markers of maturation.
[0069] The invention is also directed to a drug delivery system
comprising decellularized cardiac extracellular matrix for
delivering cells, drugs, molecules, or proteins into a subject for
treating defective, diseased, damaged or ischemic tissues or
organs. In one embodiment, the inventive biocompatible material
comprising the decellularized cardiac extracellular matrix alone or
in combination with other components is used as a non-destructive
conduction block for treatment of arrhythmias. Therefore, the
inventive biocompatible material can be used to transplant cells,
or injected alone to recruit native cells or other cytokines
endogenous therapeutic agents, or act as a exogenous therapeutic
agent delivery vehicle.
[0070] The composition of the invention can further comprise cells,
drugs, proteins, or other biological material such as, but not
limited to, erythropoietin (EPO), stem cell factor (SCF), vascular
endothelial growth factor (VEGF), transforming growth factor (TGF),
fibroblast growth factor (FGF), epidermal growth factor (EGF),
cartilage growth factor (CGF), nerve growth factor (NGF),
keratinocyte growth factor (KGF), skeletal growth factor (SGF),
osteoblast-derived growth factor (BDGF), hepatocyte growth factor
(HGF), insulin-like growth factor (IGF), cytokine growth factor
(CGF), stem cell factor (SCF), platelet-derived growth factor
(PDGF), endothelial cell growth supplement (EGGS), colony
stimulating factor (CSF), growth differentiation factor (GDF),
integrin modulating factor (IMF), calmodulin (CaM), thymidinc
kinase (TK), tumor necrosis factor (TNF), growth hormone (GH), bone
morphogenic proteins (BMP), matrix metalloproteinase (MMP), tissue
inhibitor matrix metalloproteinase (TIMP), interferon,
interleukins, cytokines, integrin, collagen, elastin, fibrillins,
fibronectin, laminin, glycosaminoglycans, hemonectin,
thrombospondin, heparan sulfate, dermantan, chondrotin sulfate
(CS), hyaluronic acid (HA), vitronectin, proteoglycans,
transferrin, cytotactin, tenascin, and lymphokines.
[0071] Tissue culture plates can be coated with either a soluble
ligand or gel form of the extracellular matrix of the invention, or
an adsorbed form of the extracellular matrix of the invention, to
culture cardiomyocytes or other cell types relevant to cardiac
repair. This can be used as a research reagent for growing these
cells or as a clinical reagent for culturing the cells prior to
implantation. The extracellular matrix reagent can be combined with
other tissue matrices and cells.
[0072] For gel reagent compositions, the solution is then
neutralized and brought up to the appropriate concentration using
PBS/saline or other buffer, and then be placed into tissue culture
plates and/or wells. Once placed in an incubator at 37.degree. C.,
the solution forms a gel that can be used for any 2D or 3D culture
substrate for cell culture. In one embodiment, the gel composition
can be crosslinked with glutaraldehye, formaldehyde, bis-NHS
molecules, or other crosslinkers, or be combined with cells,
peptides, proteins, DNA, drugs, nutrients, survival promoting
additives, proteoglycans, and/or glycosaminolycans, or combined
and/or crosslinked with a synthetic polymer for further use.
[0073] The invention further provides an exemplary method of
culturing cells adsorbed on a decellularized cardiac extracellular
matrix comprising the steps of: (a) providing a solution comprising
the biocompatible material of decellularized ECM in low pH
solution, including but not limited to, 0.5 M, or 0.01 M acetic
acid or 0.1M HCl to a desired concentration, (b) placing said
solution into tissue culture plates or wells, (c) incubating said
tissue culture plates or wells above room temperature such as at
37.degree. C., for between 1 hour and overnight (or at room
temperature to 40.degree. C.), (d) removing excess solution, (e)
rinsing said tissue culture plates or wells with PBS, and (f)
culturing cells on the adsorbed matrix. Cells that can be cultured
on the adsorbed matrix comprising the cardiac extracellular matrix
of the invention include cardiomyocytes or other cell types
relevant to cardiac repair, including stem cells and cardiac
progenitors.
[0074] In some instances a composition comprises crosslinkers
including, but not limited to, common collagen crosslinkers,
hyaluronic acid crosslinkers, or other protein cross-linkers with
altered degradation and mechanical properties.
[0075] In an instance, a method of making the composition herein
comprises electrospinning. In some instances, a method herein is
configured to control the nanofiber size, shape, or thickness.
[0076] In some instances, contractility can be induced into the
composition, for example, with cells or external pacing.
Contractility can create cyclic stress to promote a more natural
myocardium.
[0077] In some instances, cell influx and angiogenesis can be
induced into the composition, for example, when the composition
comprises linked groups or embedded factors, such as angiogenic
factors.
[0078] In some instances, a composition herein may contain
microbeads. Microbeads can be a part of the composition or
delivered by the composition. Exemplary microbeads can be any
variety of materials, for example, natural or synthetic. In some
instances, the microbeads can have varied degradation properties or
comprise, for example, MMP inhibitors, growth factors, or small
molecules.
[0079] In some instances, the composition can comprise a biological
group that can act as an adhesive or anchor where the composition
is delivered.
[0080] In an instance, a composition can be a bioadhesive, for
example, for wound repair. In some instances, a composition herein
can be configured as a cell adherent. For example, the composition
herein can be coating or mixed with on a medical device or a
biologic that does or does not comprises cells. For example, the
composition herein can be a coating for a synthetic polymer
vascular graft. In some instances, the composition includes an
anti-bacterial or anti-bacterial agents could be included.
[0081] Methods herein can comprise delivering the composition as a
wound repair device. For example, after cardiac ablation, the
composition can be delivered to improve healing.
[0082] In an instance, a composition comprises an alginate bead
that is coated with an ECM composition as described herein.
[0083] In some instances, the composition is injectable. An
injectable composition can be, without limitation, a powder,
liquid, particles, fragments, gel, or emulsion. The injectable
composition can be injected into a heart or in many instances,
injected into the left ventricle, right ventricle, left atria,
right atria, or valves of a heart. The compositions herein can
recruit, for example without limitation, endothelial, smooth
muscle, cardiac progenitors, myofibroblasts, stem cells, and
cardiomyocytes.
[0084] Methods of making the compositions herein can include
decellularizing tissue from any age animal or human by methods well
known in the art.
[0085] In some instances, a composition herein comprises ECM and a
natural or synthetic polymer. For example, a composition herein
comprises a natural polymer such as collagen, chitosan, alginate,
glycosaminoglycans, fibrin, or hyaluronic acid. In another example,
a composition herein comprises a synthetic polymer, for example
without limitation, polyethylene glycol, poly(glycolic)acid,
polylactic acid), poly(hydroxy acids), polydioxanone,
polycaprolactone, poly(ortho esters), poly(anhydrides),
polyphosphazenes, poly(amino acids), pseudo-poly(amino acids),
conductive polymers (such as polyacetylene, polypyrrole,
polyaniline), or polyurethane or their potential copolymers. In
some instances, a composition here comprise ECM and both a natural
and a synthetic polymer. A composition herein can be a
multi-material by linking an ECM and another polymer material, for
example, via reaction with amines, free thiols, or short peptides
that can self assemble with the ECM.
[0086] Methods herein include delivery of a composition comprising
an ECM. Exemplary methods include, but are not limited to: direct
injection during surgery; direct injection through chest wall;
delivery through catheter into the myocardium through the
endocardium; delivery through coronary vessels; and delivery
through infusion balloon catheter. The composition can also be
delivered in a solid formulation, such as a graft or patch or
associated with a cellular scaffold. Dosages and frequency will
vary depending upon the needs of the patient and judgment of the
physician.
[0087] In some instances, a composition herein is a coating. The
coating can comprise an ECM from any tissue for example cardiac
muscle, skeletal muscle, pericardium, liver, adipose tissue, and
brain. A coating can be used for tissue culture applications, both
research and clinical. The coating can be used to coat, for example
without limitation, synthetic or other biologic
scaffolds/materials, or implants. In some instances, a coating is
texturized or patterned. In some instances, a method of making a
coating includes adsorption or chemical linking. A thin gel or
adsorbed coating can be formed using an ECM solution form of the
composition. In some instances, a composition herein is configured
to seal holes in the heart such as septal defects.
[0088] A composition herein can also be developed from other
tissues, such as skeletal muscle, pericardium, liver, adipose
tissue, and brain. The compositions may be used as coating for
biologics, medical devices or drug delivery devices.
[0089] The reconstruction of skeletal muscle, which is lost by
injury, tumor resection, or various myopathies, is limited by the
lack of functional substitutes. Surgical treatments, such as muscle
transplantation and transposition techniques, have had some
success; however, there still exists a need for alternative
therapies. Tissue engineering approaches offer potential new
solutions; however, current options offer incomplete regeneration.
Many naturally derived as well as synthetic materials have been
explored as scaffolds for skeletal tissue engineering, but none
offer a complex mimic of the native skeletal extracellular matrix,
which possesses important cues for cell survival, differentiation,
and migration.
[0090] The extracellular matrix consists of a complex
tissue-specific network of proteins and polysaccharides, which help
regulate cell growth, survival and differentiation. Despite the
complex nature of native ECM, in vitro cell studies traditionally
assess cell behavior on single ECM component coatings, thus posing
limitations on translating findings from in vitro cell studies to
the in vivo setting. Typically, purified matrix proteins from
various animal sources are adsorbed to cell culture substrates to
provide a protein substrate for cell attachment and to modify
cellular behavior. However, these approaches would not provide an
accurate representation of the complexity microenvironment. More
complex coatings have been used, such as a combination of single
proteins, and while these combinatorial signals have shown to
affect cell behavior, it is not as complete as in vivo. For a more
natural matrix, cell-derived matrices have been used. Matrigel is a
complex system; however, it is derived from mouse sarcoma, and does
not mimic any natural tissue. While many components of ECM are
similar, each tissue or organ has a unique composition, and a
tissue specific naturally derived source may prove to be a better
mimic of the cell microenvironment.
[0091] Skeletal muscles are composed of bundles of highly oriented
and dense muscle fibers, each a multinucleated cell derived from
myoblasts. The muscle fibers in native skeletal muscle are closely
packed together in an extracellular three-dimensional matrix to
form an organized tissue with high cell density and cellular
orientation to generate longitudinal contraction. Skeletal muscle
can develop scar tissue after injury which leads to a loss of
functionality. The engineering of muscle tissue in vitro holds
promise for the treatment of skeletal muscle defects as an
alternative to host muscle transfer. Tissue engineering
compositions must be biocompatible and capable of being
vascularised and innervated.
[0092] The extracellular matrix (ECM) consists of a complex
tissue-specific network of proteins and polysaccharides, which help
regulate cell growth, survival and differentiation. Despite the
complex nature of muscle ECM, in vitro cell studies traditionally
assess muscle cell behavior on single ECM component coatings, thus
posing limitations on translating findings from in vitro cell
studies to the in vivo setting. Overcoming this limitation is
important for cell-mediated therapies, which rely on cultured and
expanded cells retaining native cell behavior over time.
[0093] In an aspect, a composition herein comprises ECM that is
derived from from porcine skeletal and cardiac muscle. The
composition can be developed for substrate coating for a variety of
applications. In some instances, the ECM of the composition retains
a complex mixture of muscle-specific ECM components after
solubilization. In some instances, the coatings herein can more
appropriately emulate the native muscle ECM in vitro.
[0094] Skeletal myoblasts plated on skeletal muscle matrix
displayed a significant increase in i) the number of myosin heavy
chain positive myotubes, ii) the number of nuclei per myotube and
iii) myotube width when compared to cells plated on traditional
collagen type I coated substrates. Human embryonic stem cell (HES2)
derived cardiac myocytes plated on myocardial matrix also displayed
a significant increase in i) myofibrillar area, ii) number of
cardiomyocyte nuclei per myofibrillar area and iii) desmosomal
plaque size, which highlights larger more mature intercalated disc
localization of the desmosomal cell-cell junction protein,
desmoplakin, when compared to cells plated on traditional gelatin
coated substrates. In some instances, the compositions are
configured to provide the ability to reconstitute the in vivo
muscle ECM. The composition may provide a tool to assess and
maintain muscle and stem cell behavior in vitro similar to the
native state, and may provide a tool for cell-mediated therapies in
vivo.
[0095] FIG. 2 illustrates the average myofibrillar area of
cardiomyocytes was significantly greater when grown on cardiac ECM
when compared to the standard coating of gelatin. FIG. 3
illustrates the average number of cardiomyocytes was significantly
higher on cardiac ECM when compared to the standard coating of
gelatin. As illustrated in FIG. 4, desmoplakin, an intracellular
junction protein, specifically localized between cardiomyocytes and
formed organized desmosomes at day 112 on cardiac ECM, but not on
gelatin.
[0096] As described herein a skeletal muscle matrix can be created
in the same or a similar manner to the cardiac ECM. The skeletal
muscle matrix can be injected into skeletal muscle for skeletal
muscle tissue engineering. FIG. 5 illustrates skeletal myoblasts
cultured on skeletal muscle matrix as described herein that
demonstrated increased myotube size, increased differentiation, and
had more nuclei per myotube than myoblasts cultured on collagen.
Using a transwell migration assay, in vitro, skeletal myoblasts
migrate specifically towards skeletal muscle matrix as illustrated
in FIG. 6.
[0097] The invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. It is apparent for skilled
artisans that various modifications and changes are possible and
are contemplated within the scope of the current invention.
EXAMPLE 1
[0098] Various studies to treat MI have investigated the injection
of cells directly into the infarct wall, although many studies have
shown poor survival rates. The objective of this study is to
examine the use of a gel as a growth platform for cell adhesion,
growth, maturation, and delivery in vivo. It is provided that a gel
composed of native heart extracellular matrix tissue can aid in
cardiac tissue regeneration by promoting cell survival.
[0099] Female Sprague Dawley rats were enthanized and their hearts
decellularized using a procedure modified from Ott et al. (Nature
Medicine, 14(2), 213, 2008). Decellularized hearts were then
lyophilized, rehydrated, pulverized, and lyophilized again to form
a dry powder. The ECM was then minimally digested in pepsin and
neutralized, as modified from Freytes et al. (Biomaterials 29:
1630, 2008).
[0100] More specifically, adult female Sprague Dawley rats were
heparinized and anesthetized intraperitoneally with pentobarbital.
The aorta and pulmonary artery were transected and the heart was
removed. The aorta was cannulized and attached to a modified
Langendorff setup.
[0101] The heart was decellularized using a modified, previously
published technique. Briefly, the coronary vessels of the heart
were retrogradedly perfused with a 1% sodium dodecyl sulfate (SDS)
and PBS solution for 24 hours and then a 1% triton PBS solution for
30 minutes. Once the decellularization was complete, the heart was
rinsed with deionized water and freeze dried in a lyophilizer.
[0102] Frozen hearts were rehydrated with water and then immersed
in liquid nitrogen. Once frozen, hearts were systematically crushed
within a ball and cup apparatus at 70 psi for 10 seconds.
Pulverized heart particulates were then freeze dried. Once dry,
lyophilized heart tissue was combined with 1% pepsin and
amalgamated with 0.01M HCl to a concentration of 10 mg/mL. Solution
was stirred at room temperature for 48 hours to allow for
solubilization of the extracellular matrix tissue. After 48 hours,
the HCl solution was aliquoted into Eppendorf tubes on ice and
neutralized with 0.1N NaOH to pH 7.4.
[0103] Through the methods described above, a native rat cardiac
ECM gel has been formed. Successful gelation of 2.5-8 mg/mL gels
occurred within 15 minutes, as confirmed by the increased viscous
nature of the material. Increased stiffness was observed with
higher density gels.
[0104] The neutralized solution was diluted to concentration with
1.times.PBS, plated on a 96 well plate at 50 .mu.L per well, and
then transferred to an incubator at 37.degree. C. and 5% CO2. After
the gel had formed, 100 .mu.L of isolated 2d neonatal cardiomyocyte
cells were pipetted on top of the gel at 60,000 cells per well.
After a few days, cells were examined for adherence to the
gels.
[0105] After heart extracellular matrix tissue had been
decellularized, pulverized and digested, a gel formed once the
solution had been brought up to physiological conditions (pH=7.4,
37.degree. C.). Gels formed with higher concentrations of ECM
tissue in solution were stiffer and more opaque than gels formed
with weaker concentrations of ECM. Cells plated on the gels were
able to adhere to and survive on the gels.
[0106] Plating cardiomyocytes on the cardiac ECM gels at
1.times.10.sup.4 showed successful adhesion and survival of cells
to the ECM. The cells were cultured on the ECM for up to four
days.
[0107] One hundred mL of cardiac ECM solution (7 mg/mL) was
injected through a 30G needle into the LV free wall of an
anesthetized rat. The present study shows that native heart
extracellular matrix can be isolated, solubilized, and
self-assembled into a gel when brought to physiological pH and
temperature. Since the gel contains all of the native extracellular
matrix components, albeit scrambled, it is provided that this
matrix allows for successful adhesion and growth of cardiomyocytes
in vitro and also once injected in vivo. Furthermore, a gel
composed of the matrix derived originally from the heart ventricles
is believed to support cardiomyocyte growth more successfully
rather than other matrices such as collagen or fibrin gels since it
more closely mimics the in vivo cardiac environment.
[0108] An injectable gel can potentially conform to any
three-dimensional shape and improve cell transplant survival within
the heart. Injected cardiomyocytes or cell which can differentiate
into cardiomyocytes can aid in the regeneration of heart tissue,
improve cardiac output. The method developed to create a native
cardiac ECM gel platform with varied concentration and stiffness
also provides an in vitro platform for cell growth and as an in
situ engineered scaffold for generation. The native ECM provides
the appropriate complex environment when injected in vivo to
increase cell retention and promote tissue regeneration for
myocardial tissue engineering.
EXAMPLE 2
[0109] Cardiomyocytes have been typically cultured on surfaces
coated with one, or possibly a few extracellular matrix (ECM)
proteins. Yet, in vivo, cardiomyocytes exist in a highly complex
extracellular milieu; an ECM that more closely mimics this native
environment may be beneficial for cultured cardiomyocyte survival.
Here, the use of native cardiac ECM that has been solubilized as a
coating for cell culture of neonatal cardiomyocytes is
reported.
[0110] Hearts were removed from Sprague-Dawley rats and
decellularized using a modified Langendorff setup (modified from
Ott et al., 2008). The decellularized hearts were lyophilized,
rehydrated, and pulverized after freezing in liquid N2. The ECM was
minimally digested in pepsin in 0.01M HCl. After 48 hours, 0.01 M
acetic acid was added to make the final concentration of 1
mg/ml.
[0111] Cardiac myocytes were harvested from freshly dissected
ventricles of 1 to 2 day old Sprague-Dawley rats using an isolation
kit (Cellutron, Highland Park, N.J.). The initial supernatant was
discarded, but the subsequent 20 min digestions were strained and
suspended in DMEM supplemented with 17% M199, 10% horse serum, 5%
fetal bovine serum, and 1% penicillin/streptomycin. After
isolation, the supernatant was pre-plated onto tissue culture
polystyrene dishes to increase purity of cardiomyocytes through
selective adhesion of fibroblasts.
[0112] Either 1 mg/ml native cardiac ECM or Collagen I (Sigma, St.
Louis, Mo.) was adsorbed onto glass coverslips for one hour at
37.degree. C. Isolated neonatal cardiomyocytes were plated at a
density of 200,000/cm.sup.2 and media was changed to low serum
maintenance after 24 hours (DMEM, 18.5% M199, 5% HS, 1% FBS and
antibiotics). Cell cultures were maintained at 37.degree. C. and 5%
CO2, monitored daily, and fresh maintenance media was exchanged
every 2-3 days.
[0113] Cardiomyocytes adhered to the adsorbed native ECM, and
formed a partially confluent layer. Initially, the cardiomyocytes
adhered at a similar density to the collagen coating.
[0114] Both cell cultures began to spontaneously beat on Day 3
after plating. Cardiomyocytes cultured on collagen began to detach
on Day 12, and stopped beating at Day 14. However, the
cardiomyocytes cultured on the native heart ECM formed clearly
defined fibrils, which beat at the same rate up until Day 28.
[0115] This study demonstrated that the use of native heart ECM for
culture of cardiomyocytes is useful as it more closely mimics the
conditions in vivo. The study also provides that neonatal
cardiomyocytes adhere and continue to function longer on the native
cardiac ECM than on the typical collagen coating. This new surface
coating is beneficial for the culture of stem cell derived
cardiomyocytes as well as cardiac progenitors.
EXAMPLE 3
[0116] Here, cell coating use has been investigated for native
heart extracellular matrix of adult ventricles that have been
decellularized and solubilized. The advantages being that native
heart ECM may have more components than traditional cell coatings,
and be more readily available for use than pretreatment with other
cell types.
[0117] Hearts were removed from Sprague-Dawley rats, and
decellularized using a modified Langendorff setup (modified from
Ott et al, 2008). The decellularized hearts were lyophilized,
rehydrated, and pulverized after freezing in liquid nitrogen. The
ECM was then digested in pepsin in 0.1M HCl. After 48 hours of
digestion, 0.01 M acetic acid was added to dilute to the final
concentration of 1 mg/ml.
[0118] Pepsin digestion of the native heart ECM was run in vertical
gel electrophoresis in reducing conditions using DTT and compared
against laminin (BD Biosciences), and calf skin collagen (Sigma).
Gels were stained with Imperial Protein Stain (Pierce). Native
heart ECM can demonstrate a more complex mixture of ECM components
when compared to collagen and laminin.
[0119] Cardiac myocytcs were harvested from the ventricles of 1 to
2 day old Sprague-Dawley rats using an isolation kit (Cellutron,
Highland Park, N.J.). The initial supernatant was discarded, but
the subsequent 20 min digestions were strained and suspended in
DMEM supplemented with 17% M199, 10% horse serum, 5% fetal bovine
serum, and 1% penicillin/streptomycin. After isolation, the
supernatant was pre-plated onto tissue culture polystyrene dishes
to increase purity of cardiomyocytes through selective adhesion of
fibroblasts.
[0120] Either 1 mg/ml native cardiac ECM or Collagen I (Sigma, St.
Louis, Mo.) was adsorbed onto tissue culture 48-well plates for 1
hour at 37.degree. C. Isolated neonatal cardiomyocytes were plated
at a density of 200,000/cm2 and media was changed to low serum
maintenance media after 24 hours (DMEM, 18.5% M199, 5% HS, 1% FBS
and 1% penicillin/streptomycin). Cell cultures were maintained at
37.degree. C. and 5% carbon dioxide, monitored daily, and fresh
media was added every 2-3 days. Cultures were fixed at day 2, day
4, and day 7 and stained for alpha actinin, connexin43,
pan-cadherin, actin and nuclei. Cardiomyocytes began to
spontaneously beat in culture at Day 2. Cells cultured on collagen
began detaching from the plate at Day 8. One set of cells cultured
on native heart ECM continued beating until Day 45. All cells
cultured on collagen stopped beating at Day 14.
[0121] Current cell culture coatings are generally simple proteins
adsorbed onto tissue culture plates or scaffolds. Using a more
complex environment is beneficial for cell survival and maturation.
The native cardiac ECM was shown by this study to contain more
complex components when compared to other standard cell culture
coatings. Neonatal rat cardiomyocytes attached to native heart ECM
as a coating for cell culture, and spontaneously began beating.
Cardiomyocytes cultured on native cardiac ECM demonstrated
increased actinin, connexin43, and pan-cadherin staining over time.
Also, the neonatal cardiomyocytes had increased survivability and
attachment on the native heart ECM when compared to collagen.
EXAMPLE 4
[0122] Here, the use of a gel as described herein is investigated
wherein the gel is made from native decellularized heart ECM. The
gel may act as an in situ gelling scaffold, providing a natural
cardiac matrix to improve cell retention and survival in the LV
wall.
[0123] Female Sprague Dawley rats hearts and porcine hearts have
been decellularized. Cardiac tissue was sliced to be .about.2 mm
thick and was rinsed with deionized water, then stirred in 1%
sodium dodecyl sulfate (SDS) until decellularized, 4-5 days. An
additional stir step in 1% Triton X-100 for 30 minutes ensured
complete decellularization and was followed by overnight stirring
in deionized water and a final rinse in deionized water.
[0124] Decellularized hearts were then lyophilized, pulverized or
milled, and lyophilized again to fonn a dry powder. The ECM was
then digested in pepsin and neutralized.
[0125] Solubilized cardiac ECM was then brought to physiologic or
pH 8, through the addition of sodium hydroxide and 10.times.PBS.
Neutralized cardiac extracellular matrix solution was then diluted
with PBS to the desired concentration and allowed to gel in 96 well
plates at 37.degree. C. Successful gelation of 2.5-8 mg/mL gels was
confirmed by visual inspection of the material. Increased stiffness
was observed with higher concentration gels.
[0126] Various experimental conditions were tested to determine
different digestion for gelation of cardiac ECM scaffolds. Vertical
gel electrophoresis was performed to compare the content of
digestion conditions, and to compare ECM content to rat tail
collagen. Initial pH was determined to play an important role in
digestion and gelation of cardiac ECM. Digestions were performed
for 48-72 hours.
[0127] Gel electrophoresis reveals an incomplete digestion of
native cardiac ECM by 0.01M HCl. Digestions of cardiac ECM in 0.1M
HCl showed increased degradation. Thus, stronger acidic conditions
were shown to improve digestion and gelation of cardiac ECM
solutions. Comparison of the cardiac ECM to rat tail collagen
demonstrates the presence of various additional peptides in the
cardiac ECM.
[0128] Scanning electron microscopy was used to visualize the
structure of the cardiac extracellular matrix gel form. Gels were
fixed with 2.5% gluteraldehyde for 2 hours, followed by a series of
ethanol rinses (30-100%), and critically point dried. Samples were
sputter coated with chromium prior to imaging.
[0129] Solubilized native ECM at a concentration of 6 mg/mL cardiac
ECM was successfully injected through a 30G needle into rat LV free
wall, creating an in situ gelled scaffold, to which cardiomyocytes
adhere and proliferate.
EXAMPLE 5
[0130] In vitro chemoattractive properties of the cardiac
decellularized ECM solution were tested using a commercially
available migration assay kit. Briefly, human coronary artery
endothelial cells (HCAECs) and rat aortic smooth muscle cells
(RASMCs) were serum starved and migration was evaluated towards the
matrix, collagen, pepsin, and fetal bovine serum. RASMCs show
significant migration towards the matrix, while HCAECs show a
trend. Thus, biochemical cues of the matrix have chemoattractive
properties that could promote cell infiltration in vivo.
[0131] In vivo, arteriole formation was quantified within the
injected region to assess neovascular formation. Arteriole density
was significantly greater at 11 days post injection, as compared to
4 hours post injection.
EXAMPLE 6
[0132] Several cell types have been shown to preserve cardiac
function when injected into the myocardial wall following an MI.
However, an acellular treatment could eliminate the complications
of poor cell survival and the immune response, common with cell
therapies.
[0133] Myocardial infarction was induced in rats using a 25 min
ischemia-reperfusion model, via occlusion of the left anterior
descending artery. At one week post-MI baseline function was
calculated from MRI images.
[0134] Porcine myocardial ECM was decellularized in small pieces,
in 1% SDS for several days, followed by a DI rinse overnight,
lyophilization and milling to create a powder. Digestion was
performed in 0.1 M HCl with pepsin to create a solubilized form of
the material.
[0135] Solubilized ECM was brought to pH 7.4 using 1 M NaOH and
diluted with PBS to be 6 mg/mL prior to injection. After MI
surgery, animals were randomized into two groups and ECM or saline
was injected into the LV free wall of female Sprague dawley rats
through a 30 G needle, two weeks after infarction surgery.
[0136] 4 weeks after injection surgery (6 weeks post-MI), cardiac
function was again assessed using MRI.
[0137] Animals injected with ECM showed preserved function (as
evaluated based on ejection fraction) at 6 weeks, while saline
injected animals did not maintain cardiac function. End diastolic
and end systolic volume were also preserved in ECM injected
animals.
EXAMPLE 7
[0138] Currently, stem cells and other cell types are in clinical
trials for treatment of heart failure by delivery through a 27 G
catheter into the myocardial wall. Porcine ventricular tissue was
decellularized using SDS detergents, and processed to form a
solubilized form of the matrix, and neutralized to physiologic pH
and diluted to 6 mg/mL for injection.
[0139] Two Yorkshire pigs received a coil-induced myocardial
infarction and were injected with myocardial matrix alone or with
cells at two months post infarction.
[0140] Derived from fetal cardiac explants were pre-labeled with
DiI, a cyotoplasmic stain, for histological identification. A
pro-survival cocktail, shown to enhance hESC survival in a rodent
model, was used.
[0141] Matrix alone or with cells was injected at a clinically
relevant rate of 0.2 mL per 30 seconds through a catheter, as
guided by NOGA mapping. 5 injections of 0.1 mL each were made of
matrix alone or with cells into border zone regions of the
infarct.
[0142] Matrix alone and matrix with cells was able to be
successfully injected into the porcine heart, minimally invasively,
without clogging the thin catheter.
EXAMPLE 8
[0143] Here, the investigation and use of a gel derived from
decellularized pericardial tissue is described as pertaining to its
potential as an autologous therapy to improve cell retention and
survival in the LV wall by promoting neovascularization in
vivo.
[0144] Both porcine and human pericardia have been decellularized.
Juvenile male farm pigs were euthanized and their pericardia were
decellularized via procedures modified from Ott et al. (Nature
Medicine, 14(2), 213, 2008). Specifically, pericardia were rinsed
briefly in DI water, stirred in 1% sodium dodecyl sulfate (SDS) for
24 hours, then stirred in DI water for approximately 5 hours. Human
pericardial tissue samples were collected from patients undergoing
cardiothoracic surgeries. These samples were decellularized in a
similar manner: a brief DI rinse, followed by 3 days in 1% SDS,
followed by an overnight DI rinse. In both cases, complete
decellularization was verified with histological staining.
[0145] The following is valid for both human and porcine
pericardial ECM samples.
[0146] Decellularized pericardia, or pericardial ECM, were then
frozen, lyophilized, and milled to form a fine, dry powder. The ECM
powder was then digested with pepsin dissolved in HCl and
neutralized, via methods modified from Freytes et al. (Biomaterials
29: 1630, 2008).
[0147] Gel electrophoresis (SDS-PAGE) indicated greater complexity
than in pepsin-digested collagen, showing a wide range of smaller
bands in the pericardium samples.
[0148] This complexity was confirmed by analyzing the samples with
mass spectroscopy to identify protein fragments. Fragments of ECM
proteins identified included collagen, elastin, fibrin, and a
variety of proteoglycans.
[0149] When 150 ul of the neutralized solution was loaded into a
96-well plate and allowed to stand in an incubator, gelation was
observed after 2-3 hours.
[0150] In vivo gelation was observed by injecting 60 ul of the
neutralized ECM solution into the left ventricular (LV) wall of
male Sprague Dawley rats. Histological staining of hearts sectioned
from animals sacrificed 45 minutes after injection showed an area
of gelled ECM visible in the LV wall.
[0151] In the same experiment, animals were maintained for two
weeks, after which they were sacrificed and their hearts were
harvested for sectioning. The ECM injection was still visible at
this time point, but had been infiltrated by cells.
[0152] Immunohistochemistry was performed on tissue slices in order
to identify the smooth muscle cells and endothelial cells,
indicative of blood vessels. The presence of a large number of
vessels within the ECM injection area indicates that the material
promotes neovascularization.
EXAMPLE 9
[0153] Surface coatings for in vitro cell culture have been
typically made of one or a few extracellular matrix proteins. While
this provides a cell adhesive surface, it does not mimic the in
vivo extracellular microenvironment. Herein was developed a method
to generate adsorbed coatings derived from extracellular matrices
of various tissues, including cardiac, skeletal muscle, liver,
pericardium, adipose tissue, and brain.
[0154] Tissue from porcine and rat origin was taken and
decellularized. Cardiac tissue, skeletal muscle, and liver of both
rat and porcine origin and brain, fat and pericardium of porcine
origin was decellularized using various detergents. Cardiac,
skeletal muscle, and liver tissue was sliced to be .about.2 mm
thick and was rinsed with deionized water, then stirred in 1%
sodium dodecyl sulfate (SDS) in PBS until decellularized. The time
it took to decellularize depended on tissue type. Brains were cut
in half and stirred slowly in 0.001% SDS in PBS. Pericardial tissue
was decellularized in 1% SBS in PBS, and adipose tissue was
decellularized in 2.5 mM sodium deoxycholate, and then further
processed with lipase. Other decellularization agents have also
been tested. Decellularized tissue was then rinsed in deionized
water to ensure removal of detergents, and then lyophilized.
[0155] The decellularized ECM was milled to form a dry powder, with
the exception of decellularized brain and adipose ECM. The ECM was
then digested to form a solubilized form used as a coating using
pepsin in low acid conditions and then diluted using 0.1M acetic
acid to bring it to the desired concentration of 1 mg/ml. Vertical
Polyacrylamide Gel Electrophoresis was used and demonstrated a
complex mixture of peptide fragments in each tissue type, which
varied from tissue to tissue. This demonstrates that there exists
tissue specific components in the decellularized ECM.
[0156] These coatings can be applied to surfaces in the same manner
as typical single protein coatings. By culturing cells on tissue
specific coatings that mimic the in vivo extracellular matrix
microenvironment, there was better control of survival and cell
morphology, and enhance differentiation.
[0157] Rat cortical neurons were cultured on brain matrix and
compared to a standard coating of poly-1-lysine. Rat cortical
neurons survived and retained their branched morphology longer on a
brain matrix coating compared to the standard coating. Also
observed was increased percent differentiation and increased
myotube width when skeletal myoblasts were cultured on a skeletal
muscle matrix coating compared to the standard coating of collagen.
Finally, human embryonic stem cell derived cardiomyocytes displayed
increased organization and maturation, including the formation
cell-cell junctions when plated on a cardiac matrix coating
compared to the typical gelatin coating.
[0158] These studies indicate the importance of using extracellular
matrix mimics for cell culture, with implications towards many in
vitro cell studies, including the promotion of stem cell maturation
and differentiation.
EXAMPLE 10
[0159] Here, the use of the gel is described herein is investigated
wherein the gel is made from native decellularized skeletal muscle
ECM. The gel can act as an in situ gelling scaffold, providing a
natural skeletal muscle matrix to improve tissue regeneration in a
leg injury model. The advantage is that the skeletal muscle ECM has
components similar to the matrix found in vivo, and may provide a
suitable platform for tissue engineering and regeneration, cell
recruitment, and cell delivery.
[0160] Porcine skeletal muscle was decellularized. The tissue was
sliced to be .about.2 mm thick and was rinsed with deionized water,
then stirred in 1% sodium dodecyl sulfate (SDS) in PBS until
decellularized. Decellularized tissue was then rinsed in deionized
water to ensure removal of detergents. Pieces of decellularized
tissue were sectioned and stained using hematoxylin and eosin to
ensure removal of cells. Decellularized tissue was then lyophilized
and milled to form a fine powder.
[0161] The skeletal muscle ECM was then digested in pepsin in low
acid conditions, and then neutralized to physiologic or near
physiologic pH through the addition of sodium hydroxide and
10.times.PBS. Neutralized skeletal muscle ECM solution was then
diluted with PBS to the desired concentration of 6 mg/ml and
allowed to gel in 96 well plates at 37.degree. C. Successful
gelation was confirmed by visual inspection of the material.
[0162] Solubilized native skeletal muscle ECM at a concentration of
6 mg/ml was successfully injected through a 25G needle into rat leg
femoral muscle creating a gelled scaffold. Gelation occurred within
10-15 minutes. Muscle and ECM was excised and sectioned and stained
using hematoxylin and eosin to confirm successful gelation of
skeletal muscle ECM in the muscle.
[0163] Skeletal muscle ECM can also be used to deliver cells, such
as skeletal myoblast or other muscle relevant cell types in the
ECM.
[0164] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *