U.S. patent application number 16/123156 was filed with the patent office on 2019-02-07 for energetic three-dimensional artificial cardiac patch and uses thereof.
This patent application is currently assigned to University of Houston. The applicant listed for this patent is University of Houston. Invention is credited to Ravi K. Birla.
Application Number | 20190040360 16/123156 |
Document ID | / |
Family ID | 51841526 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190040360 |
Kind Code |
A1 |
Birla; Ravi K. |
February 7, 2019 |
ENERGETIC THREE-DIMENSIONAL ARTIFICIAL CARDIAC PATCH AND USES
THEREOF
Abstract
In some embodiments, the present disclosure provides a method
for fabricating a three-dimensional artificial cardiac patch
construct. In some embodiments, such method includes the steps of
coating a substrate with an organic polymer; allowing the organic
polymer coating to air dry; mounting anchors on the organic polymer
coating; and sterilizing the organic polymer coating and the
anchors. In further embodiments, the method includes the steps of
forming a biodegradable gel-based support scaffold on top of the
organic polymer coating and seeding the biodegradable gel-based
support scaffold with neonatal cardiac cells. In yet further
embodiments, the method comprises culturing the neonatal cardiac
cells in vitro to form a real cardiac layer, under culture
conditions that are suitable for the cells to self-organize into a
monolayer and detach from the substrate to form the
three-dimensional cardiac patch. In some embodiments, the present
disclosure pertains to a method of treatment of cardiac tissue
injury in a subject in need thereof. In some embodiments, the
method includes implanting the three-dimensional artificial cardiac
patch described above in the injured area of the subject. In
another embodiment the present disclosure provides a composition
comprising the three-dimensional artificial cardiac patch described
above. Additional embodiments of the present disclosure pertain to
a medicament including the three-dimensional artificial cardiac
patch described above.
Inventors: |
Birla; Ravi K.; (Sugar Land,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Houston |
Houston |
TX |
US |
|
|
Assignee: |
University of Houston
Houston
TX
|
Family ID: |
51841526 |
Appl. No.: |
16/123156 |
Filed: |
September 6, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14270766 |
May 6, 2014 |
10106776 |
|
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16123156 |
|
|
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61819843 |
May 6, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0657 20130101;
A61K 35/34 20130101; A61L 27/52 20130101; C12N 2533/56 20130101;
A61K 38/18 20130101; A61L 2430/20 20130101; A61L 27/225 20130101;
A61L 27/34 20130101; C12N 2533/30 20130101; A61L 27/3826 20130101;
A61L 27/34 20130101; C08L 83/04 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; A61K 38/18 20060101 A61K038/18; A61L 27/52 20060101
A61L027/52; A61K 35/34 20060101 A61K035/34; A61L 27/34 20060101
A61L027/34; A61L 27/22 20060101 A61L027/22; A61L 27/38 20060101
A61L027/38; C08L 83/04 20060101 C08L083/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0002] This invention was made, at least in part, with U.S.
government support under grant No. RO1-EB011516 awarded by the
National Institute of Health. The U.S. government may have certain
rights in this invention.
Claims
1. A method for the treatment of cardiac tissue injury comprising:
implanting a fabricated three-dimensional artificial cardiac patch
in a subject in need thereof, wherein the fabrication of the
three-dimensional artificial cardiac patch comprises: coating a
substrate with an organic polymer; allowing the organic polymer
coating to air dry; mounting anchors on the organic polymer
coating; sterilizing the organic polymer coating and the anchors;
forming a biodegradable gel-based support scaffold on top of the
organic polymer coating; seeding the biological support scaffold
with neonatal cardiac cells; and culturing the neonatal cardiac
cells in vitro to form a real cardiac layer, wherein the culture
conditions are suitable for the cells to self-organize and detach
from the substrate to form the three-dimensional cardiac patch.
2. The method of claim 1, wherein the cardiac tissue injury is due
to acute or chronic stress, atheromatous disorders of blood
vessels, ischemia, myocardial infarction, inflammatory disease and
cardiomyopathies or myocarditis.
3. The method of claim 2, wherein the acute or chronic stress is
due to systemic hypertension, pulmonary hypertension or valve
dysfunction.
4. The method of claim 2, wherein the atheromatous disorder of
blood vessels is coronary artery disease.
5. The method of claim 1, wherein the anchors are secured to the
substrate, and wherein the anchors are utilized to define outer
perimeters of the three-dimensional cardiac patch.
6. The method of claim 5 further comprising the step of trimming
the real cardiac layer around the outer perimeters defined by the
anchors, wherein the real cardiac layer detaches from the substrate
to form the three-dimensional cardiac patch.
7. A three-dimensional artificial cardiac patch comprising: an
organic polymer coated on a substrate; a biodegradable gel-based
support scaffold formed on top of the organic polymer; and cardiac
cells, wherein the cardiac cells are seeded on the biodegradable
gel-based scaffold.
8. The three-dimensional artificial cardiac patch of claim 7
further comprising at least one agent from the group consisting of
survival factors, growth factors, pharmacological agents,
angiogenic factors, beta-blockers or ACE inhibitors.
9. The three-dimensional artificial cardiac patch of claim 7,
wherein the patch is implanted in a suitable recipient.
10. The three-dimensional artificial cardiac patch of claim 9,
wherein the recipient suffers from a cardiac tissue injury or a
congenital heart disease.
11. The three-dimensional artificial cardiac patch of claim 7,
wherein the organic polymer is a silicone elastomer.
12. The three-dimensional artificial cardiac patch of claim 7,
wherein the silicone elastomer is polydimethylsiloxane elastomer
(PDMS).
13. The three-dimensional artificial cardiac patch of claim 7,
wherein the biodegradable gel-based support scaffold is
biocompatible and non-immunogenic.
14. The three-dimensional artificial cardiac patch of claim 7,
wherein the biodegradable gel-based support scaffold is fibrin.
15. The three-dimensional artificial cardiac patch of claim 7,
wherein the seeding of the biodegradable gel-based support scaffold
comprises layering the cardiac cells onto the scaffold.
16. The three-dimensional artificial cardiac patch of claim 7,
wherein the seeding of the biodegradable gel-based support scaffold
comprises embedding the cardiac cells into the biodegradable
gel-based support scaffold.
17. The three-dimensional artificial cardiac patch of claim 7,
wherein the cardiac cells comprise neonatal cardiac cells.
18. The three-dimensional artificial cardiac patch of claim 17,
wherein the neonatal cardiac cells comprise fibroblasts,
cardiomyocytes, endothelial cells, smooth muscle cells and cardiac
stem cells.
19. The three-dimensional artificial cardiac patch of claim 7,
wherein the artificial three-dimensional patch is spontaneously
contractile.
20. The three-dimensional artificial cardiac patch of claim 7,
wherein the fabricated artificial three-dimensional patch exhibits
contractile twitch force.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/270,766 filed May 6, 2014, which claims priority to
U.S. Provisional Patent Application No. 61/819,843, filed on May 6,
2013. The entirety of the aforementioned applications is
incorporated herein by reference.
BACKGROUND
[0003] Heart transplantation has been the most successful modality
in the treatment of severe Chronic Heart Failure. However,
widespread applicability is limited by the chronic shortage of
donor organs. Engineered cardiac tissues, which embed enough cells
and provide additional tension support, possess a tremendous
potential in treating large injured areas of the heart and in
replacing congenital defects of the heart. So far, prior art
methods for reconstruction of a functional heart tissue have been
fraught with problems. In particular, problems with vascularization
of the construct still limit the use of conventional tissue
scaffolds in the replacement of large-sized tissue defects.
Additionally, reproducing the special organizational, mechanical
and elastic properties of native myocardium represents a
significant challenge from the perspective of tissue engineering
scaffolds. Thus, there exists a need to have engineered cardiac
tissues that display functional and morphological properties of
native myocardium and remain viable after implantation.
SUMMARY
[0004] In some embodiments, the present disclosure provides a
method for fabricating a three-dimensional artificial cardiac patch
construct. In some embodiments, such method includes the steps of
coating a substrate with an organic polymer; allowing the organic
polymer coating to air dry; mounting anchors on the organic polymer
coating; and sterilizing the organic polymer coating. In further
embodiments, the method includes forming a biodegradable gel-based
support scaffold on top of the organic polymer coating; and seeding
the biological support scaffold with neonatal cardiac cells. In yet
further embodiments; the method comprises culturing the neonatal
cardiac cells in vitro to form a real cardiac layer, under culture
conditions that are suitable for the cells to self-organize into a
monolayer and detach from the substrate to form the
three-dimensional cardiac patch construct.
[0005] In some embodiments, the present disclosure pertains to a
method of treatment of cardiac tissue injury in a subject in need
thereof. In some embodiments, the method includes implanting the
aforementioned three-dimensional artificial cardiac patch in the
injured area of the subject.
[0006] In yet another embodiment, the present disclosure provides a
three-dimensional artificial cardiac patch comprising an organic
polymer coated on a substrate; a biodegradable gel-based support
scaffold formed on top of the organic polymer; and cardiac cells,
wherein the cardiac cells are seeded on the biodegradable gel-based
scaffold. In an embodiment, the three-dimensional artificial
cardiac patch further comprises at least one agent from the group
consisting of survival factors, growth factors, pharmacological
agents, angiogenic factors, beta-blockers or ACE inhibitors.
[0007] Additional embodiments of the present disclosure pertain to
a medicament comprising the aforementioned composition. In some
embodiments the medicament further comprises at least one agent
from the group consisting of survival factors, growth factors,
pharmacological agents, angiogenic factors, beta-blockers or ACE
inhibitors.
[0008] The above objects and other objects, features, and
advantages of the present invention are readily apparent from the
following detailed description of the best mode for carrying out
the invention when taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0009] In order that the manner in which the above recited and
other advantages and objects of the invention are obtained, a more
particular description of the invention briefly described above
will be rendered by reference to specific embodiments thereof,
which are illustrated in the appended Figures. Understanding that
these Figures depict only typical embodiments of the invention and
are therefore not to be considered limiting of its scope, the
invention will be described with additional specificity and detail
through the use of the accompanying Figures in which:
[0010] FIGS. 1A-1K depict the schematics of methods for fabrication
of the three-dimensional artificial cardiac patch construct. FIG.
1A shows coating a substrate with an organic polymer; FIG. 1B shows
allowing the organic polymer coating to air dry; FIG. 1C mounting
anchors on the organic polymer coating; FIG. 1D shows forming a
biodegradable gel-based support scaffold on top of the organic
polymer coating; FIG. 1E shows seeding the biological support
scaffold with neonatal cardiac cells; FIG. 1F shows detachment of
the gel; FIG. 1G further compacting of the gel; FIG. 1H shows
contraction direction; FIG. 1I shows shape formation of the patch;
FIG. 1J shows detection of ECG signals; and FIG. 1K shows
measurement of the twitch force.
[0011] FIG. 2 shows the effect of using different cell densities
over time on the formation of the three-dimensional artificial
patch construct. The tissue begins to detach at 4 days for the 5M
patch.
[0012] FIG. 3 shows the effect of using different cell densities
over time, on the contraction frequency of the three-dimensional
artificial cardiac patch. Contraction rates (bpm) measured and
averaged for 1 to 6M density patches at 2, 4, 6, and 8 days.
[0013] FIGS. 4A-4G depict the relationship between the density of
the cells initially seeded in the biodegradable gel-based support
scaffold and contractile force. FIG. 4A is a representative graph
depicting the steady contraction of the patches fabricated with
cell densities ranging from 1M to 3M; representative sample of high
and low rate contractile force from 2M (FIG. 4B), 4M (FIG. 4C), and
6M (FIG. 4D) patches; FIG. 4E, is a graph of representative average
contractile forces from 1M to 3M patches at various pretensions;
Graphs of average contractile force for cell densities of 1M, 2M,
3M, 4M, 5M and 6M loaded with a pretension range of 1000 to 2000
.mu.N from high rate (FIG. 4F), and low rate (FIG. 4G)
contractions.
[0014] FIGS. 5A-5E show contractile force generated by the
fabricated three-dimensional artificial cardiac patch as a function
of cell density. FIGS. 5A-5C illustrate the representative of the
continual and singular contractile forces for cell densities 2M
(FIG. 5A), 4M (FIG. 5B) and 6M (FIG. 5C). FIG. 5D represents the
graph for average contractile forces from continual contractions.
FIG. 5E shows the graph for average contractile forces from
singular contraction for cell densities 1M, 2M, 3M, 4M, 5M, and
6M.
[0015] FIGS. 6A-6I show the patch morphology of the
three-dimensional artificial cardiac patch at different
magnifications. FIG. 6A (100.times.) cross-section, FIG. 6B
(100.times.), and FIG. 6C (400.times.) planar sections directly
from frozen samples illustrate the composition of the sample patch
obtained with light microscope; Arrow heads (FIG. 6A) indicate the
real cardiac layer.
[0016] FIG. 6D (200.times.) cross-section, FIG. 6E (200.times.) and
FIG. 6F (400.times.) image from Masson trichrome; Arrow heads in
FIG. 6D indicate the real cardiac layer, and arrows in FIG. 6E is
the fibrin gel network underneath the real cardiac layer; FIG. 6G
is a planar image showing growth of heart muscle (.alpha.-Actinin),
endothelial cells (vWF) and nuclear division (Ki67), the arrows
indicate a nucleus in karyokinesis (FIG. 6H); FIG. 6I is a planar
image showing gap junction protein (Connexin 43) and endothelial
cells (vWF) in the cultured tissue. FIGS. 7A-7Q illustrate the real
cardiac layer thickness and gap junctions in the patch. FIGS. 7A-7C
are cross-sections showing real cardiac layer thickness and the
support scaffold fibrin network in the patch by Masson's trichrome
staining for cell densities of 2 M (FIG. 7A), 4M (FIG. 7B), and 6M
(FIG. 7C), respectively; FIGS. 7D-7F are cross-sections showing
growth of heart muscles (.alpha.-Actinin) and gap junctions (Cx43)
for 2 M ((FIG. 7D), 4M (FIG. 7E) and 6M (FIG. 7F) patches,
respectively; FIGS. 7G, 7J and 7M show the total signal volumes,
FIGS. 7H, 7K and 7N show the signal volumes of Cx43, and 7I, 7K and
7O show the signal volumes of (.alpha.-Actinin for 2M, 4M and 6M
patches, respectively; FIG. 7P is a graph showing differences in
patch thickness; and FIG. 7Q depicts the signal volume indexes of
Cx43 for 2M, 4M and 6M patches.
[0017] FIG. 8 is a representation of a movie from 6M patches at day
3, taken inside the incubator (448 bpm), under the microscope (448
bpm) and then on the bench.
DETAILED DESCRIPTION
[0018] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only, and are not restrictive of the invention, as
claimed. In this application, the use of the singular includes the
plural, the word "a" or "an" means "at least one", and the use of
"or" means "and/or", unless specifically stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting. Also,
terms such as "element" or "component" encompass both elements or
components comprising one unit and elements or components that
comprise more than one unit unless specifically stated otherwise.
Parameters disclosed herein (e.g., temperature, time,
concentration, etc.) may be approximate.
[0019] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated herein by reference in their entirety for any purpose.
In the event that one or more of the incorporated literature and
similar materials defines a term in a manner that contradicts the
definition of that term in this application, this application
controls.
[0020] "Angiogenesis" as used herein, generally refers to the
growth of blood vessels in the three-dimensional artificial cardiac
patch construct. The angiogenesis may occur in response to a
stimulus, for instance, in response to administration of an
effective amount of an angiogenic factor.
[0021] The term "angiogenic factor" as used herein shall be given
its ordinary meaning and shall refer to a molecule capable of
activating or otherwise promoting angiogenesis.
[0022] The term "cardiac patch" as used herein shall be given its
ordinary meaning and shall refer to tissue of the heart, for
example, the epicardium, myocardium or endocardium, or portion
thereof, of the heart.
[0023] The term "cardiac tissue injury" as used herein shall be
given its ordinary meaning and shall refer to a cardiac tissue that
is, for example, ischemic, infarcted, reperfused, or otherwise
focally or diffusely injured or diseased. Injuries associated with
a cardiac tissue include any areas of abnormal tissue in the heart,
including any areas of abnormal tissue caused by a disease,
disorder or injury and includes damage to the epicardium,
endocardium and/or myocardium. Non-limiting examples of causes of
cardiac tissue injuries include acute or chronic stress (e.g.,
systemic hypertension, pulmonary hypertension or valve
dysfunction), atheromatous disorders of blood vessels (e.g.,
coronary artery disease), ischemia, infarction, inflammatory
disease and cardiomyopathies, myocarditis or congestive heart
failure.
[0024] The term "Animal", or "Mammal," as used herein, includes
animals and humans. Thus, when referring to processes such as
harvesting tissue from an animal, it is intended that the animal
can be a human. Although at times reference may be made herein to
"an animal or human," this is not intended to imply that the term
"animal" does not include a human.
[0025] Additionally, the term as Subject" or "Recipient," as used
herein, includes individuals who require intervention or
manipulation due to a disease state, treatment regimen or
experimental design.
[0026] "Biocompatible," as used herein, generally refers to an
autologous cell or tissue that originates or is derived from the
subject or recipient.
[0027] The phrases "conditions suitable for cells to self-organize"
or "appropriate cell culture conditions" for a suitable cell type,
as used herein, generally refers to an environment with conditions
of temperature, pressure, humidity, nutrient and waste exchange,
and gas exchange that are permissive for the survival and
reproduction of the cells. With respect to any particular type of
cell, an environment suitable for growth may require the presence
of particular nutrients or growth factors needed or conducive to
the survival and/or reproduction of the cells.
[0028] "Engineered cardiac tissue construct" or "cardiac patch", or
"artificial cardiac patch construct" as used herein, generally
refers to three dimensional mass of living mammalian tissue
produced primarily by growth in vitro on a substrate. The construct
may include one or more types of cells or tissues. For example, the
construct may be made up of myocytes cultured in conjunction with
other cell types, such as endocardial cells, vascular smooth muscle
cells, vascular endothelium, fibroblast, and adrenergic cells, or
various subsets of those cell types. The term also encompasses a
three-dimensional mass of living mammalian tissue produced at least
in part by growth in vivo on a substrate. More particularly,
constructs may include two or three-dimensional tissue which share
critical structural and functional characteristics with intact
cardiac tissue, such as distinctive multicellular organization and
oriented contractile function.
[0029] "Real Cardiac Layer," as used herein, generally refers to a
self-organized monolayer of the neonatal cardiac cells and
naturally produced extracellular matrix on top of the biological
support scaffold.
[0030] As used herein, the terms "treat," "treatment" and
"treating" shall be given their ordinary meaning and shall refer to
the reduction or amelioration of the progression, severity, and/or
duration of a cardiac tissue injury or a symptom thereof. Treatment
as used herein includes, but are not limited to, preserving the
injured cardiac tissue, regenerating new cardiac tissue, increasing
blood flow to the injured tissue, increasing myocardial perfusion,
improving global cardiac function (e.g., stroke volume, ejection
fraction, and cardiac output) and regional cardiac function (e.g.,
ventricular wall thickening, segmental shortening and heart
pumping).
[0031] Tissue engineering combines cellular and molecular biology
with material and mechanical sciences to provide an alternative to
organ and tissue transplants, which face a limited supply of donor
organs. Engineered cardiac tissues, constructed with isolated cells
on a natural or synthetic scaffold, have tremendous potential to
offer alternative treatment modalities in the healing process of
large injured areas and in repairing congenital defects of the
heart. By embedding a sufficient number of cells in the tissue and
by providing additional tension support to the damaged area,
engineered cardiac tissues may circumvent low rates of cell
engraftment observed with intracoronary delivery and poor cell
survival with intramyocardial injection.
[0032] Several criteria for engineered cardiac tissue constructs or
cardiac patches have been proposed. These constructs should display
functional and morphological properties similar to native heart
muscle and remain viable after implantation. The spontaneous
contraction twitch forces, generated without any treatment, by
seeding neonatal rat heart cells on fibrin gel, exhibit a novel
natural instinct. The engineered cardiac tissue constructs of the
present disclosure, not only exhibit endothelial cell growth and
robust cellular division, but also demonstrate electromechanical
coupling protein expression, which can sustain native electrical
propagation. Furthermore, in some embodiments of the present
disclosure, it is possible to use host origin fibrinogen and
thrombin to produce nonimmunogenic fibrin scaffolds before in vivo
application.
[0033] Neonatal cardiomyocytes possess a tremendous differentiation
potential and regenerative capacity. For example, the hearts of
1-day-old neonatal mice can regenerate after a partial surgical
resection. Previous methods have utilized cardiomyocytes from 1-3
day old neonatal rats, embedded in collagen type I supplemented
with Matrigel, to fabricate a 3D heart tissue. Under optimal
supplementation, for instance culturing under auxotonic load or
with insulin, such constructs demonstrate a maximal twitch tension
of up to 2600.+-.100 .mu.N. A model for the self-organization of
primary cardiac cells on laminin substrate to form functional 3D
heart muscle, termed cardioids, which exhibited several
physiological performance metrics comparable to normal mammalian
cardiac tissue and generated twitch forces of 200-300 .mu.N by
electrically pacing at frequencies of 1-10 Hz without any signs of
fatigue, is disclosed in U.S. Patent Application No. US
2004/0132184, which is incorporated herein by reference in its
entirety.
[0034] However, the use of these conventional tissue scaffolds is
limited by lack of adequate vascularization of such constructs as
well as the challenge of reproducing the special organizational,
mechanical and elastic properties of native myocardium. The present
disclosure addresses these needs. In addition, in some embodiments
of the present disclosure, the scaffold is fabricated using a
porous fibrin gel, which supports nutrient to the cells in order to
maintain cell viability and tissue functionality.
[0035] Fibrin is a natural, self-assembling peptide found in the
body that is used to form clots along damaged endothelium. Fibrin
possesses many interesting properties, such as biocompatibility,
bioresorbability, ease of processing, ability to tailor conditions
of polymerization, and potential for incorporation of both cells
and cell mediators. Thrombin and fibrinogen, which react to form
fibrin gel, can be produced from the patient's own blood, thus
reducing the potential risk of foreign body reaction or infection
when used as component in clinical application. Native,
fully-hydrated fibrin gels form at different fibrinogen and
thrombin concentrations and at different ionic strengths. Fibrin
alone, or in combination with other materials, has been used as a
biological scaffold for stem and primary cells to regenerate
adipose tissue, bone, cardiac tissue, cartilage, liver, nervous
tissue, ocular tissue, skin, tendons, and ligaments.
[0036] In some embodiments of the present disclosure, the fibrin
support scaffold was developed with human fibrinogen and thrombin
and the cardiac cells were well incorporated into the fibrin
network. In some embodiments, a thin layer of real cardiac tissue
(FIGS. 6A, 6D) was seen on the top of fibrin gel network. In some
embodiments, the thickness of the real cardiac tissue layer varied
with the plated cell density, with the 4 million cell (4M) and 6
million cell (6M) patches having a significantly thicker layer than
the 2 million cell (2M) patches. The reason for this difference
might result from the lower cell density and/or the growth rate of
each cell type within the patch. Due to overpopulation and the
different growth rates for each cell type (fibroblasts,
cardiomyocytes, endothelial cells, smooth muscle cells and cardiac
stem cells), the cells in the uppermost portion of real cardiac
layer of 5 and 6M patches began to die and detach from day 4 (arrow
head, FIG. 2) because of the poor nutrients supply underneath.
Thus, in some embodiments of the present disclosure, in terms of
cell survivability and morphology, the optimal density for
constructing the cardiac patch may be 4 million (4M) cells.
[0037] Freshly isolated neonatal cardiac cells consist of
fibroblasts, cardiomyocytes, smooth muscle cells, endothelial cells
and cardiac stem cells. The proliferation rate of cardiac cells is
higher in the fetal stage than in the neonatal stage, and greatly
diminishes in adulthood. In an exemplary embodiment, 2 to 3-day-old
rat cardiac cells were used to construct the cardiac patch. In some
embodiments, the cardiac patch of the present disclosure stained
positive for vWF, which is secreted by endothelial cells lining a
blood vessel, thereby indicating presence of endothelial cells
(FIGS. 6G, and 6I). In further embodiments, a positive staining for
vWF in the cardiac patch indicated that there were potential
angiogenesis buds, which would be suitable to grow and connect to
host micro blood vessels and bring nutrients into the cardiac patch
during in vivo applications. Further, in some embodiments, a
positive staining for ki67 in the cardiac patch indicated that the
cardiac patch had ongoing robust cell proliferation (FIGS. 6G, 6H).
Ki67 is a nuclear and nucleolar protein which is tightly associated
with somatic cell proliferation.
[0038] In some embodiments, the cardiac patch of the present
disclosure demonstrated positive staining for the Connexin 43
(Cx43) protein, thereby indicating that cardiomyocytes in the
present cardiac tissue patch possess electromechanical coupling
(FIGS. 6I, 7D, 7F).
[0039] In some embodiments of the present disclosure, the cardiac
patches can sustain electrical propagation with speeds that would
be close to native tissues, as indicated by the detected
electrocardiogram (ECG) signal and the natural, adult-heart-like
QRS complex. The R wave amplitudes increased with thickness of real
cardiac tissue; as shown in FIGS. 5A-5C, FIG. 4E. In a preferred
embodiment, cardiac patches formed with 4M and 6M patches exhibit
greater R wave amplitude than 2M patch because they possess more
cardiomyocytes, which can generate a higher depolarization
current.
[0040] The earlier spontaneous contractions in the cardiac patches
were observed for a few of the patches with cell densities of 4M to
6M after only one day of incubation. The contraction rate at day 2
increased with higher cell densities. The average contraction rate
for a 6M patch was 430.+-.54 bpm (n=23) at day 2, which falls
within the range of a normal adult rat heartbeat. For 1M to 3M
densities, the highest mean contraction was at day 4 and then
steadily decreased from day 6 to 8. For 4M to 6M densities, the
highest contraction occurred much earlier, at day 2 but it
decreased sharply from day 4 to 8 (FIG. 3).
[0041] A synchronized contraction relies on the appropriate
proportion of cardiomyocytes to fibroblasts as well as smooth
muscle cells and endothelial cells. In an exemplary embodiment of
the present invention cardiac patches constructed with 1M to 3M
cell densities maintained the appropriate cell-type proportion that
enhanced the synchronization and increased the contraction rate.
Because the fibroblasts proliferate faster than cardiomyocytes,
after day 4 the appropriate proportion may no longer existed. The
fibroblast overpopulation, thus, may be the cause for a decrease in
contraction rate by affecting the initiation of pacemaker cells and
delaying the propagation of action potential. Thus, synchronization
slowed and the contraction rate decreased dramatically. The varying
rates of proliferation between fibroblasts and cardiomyocytes may
be a factor contributing to detection of steady contractions from
patches constructed with 1M to 3M cell densities, and arrhythmic
contractions from cardiac patch constructed from 4M to 6M cell
densities.
[0042] The strength of a muscle's contraction is influenced by the
number of fibers within the muscle that have interactions of myosin
cross bridges with actin, the rate of contraction, and the relaxed
length of the muscle fibers. In an exemplary embodiment of the
present disclosure, 1M to 3M patches exhibited steady contractions
and 4M to 6M patches arrhythmic contractions. The contractile
forces generated by 1M to 3M patches changed relative to pretension
(baseline) (FIG. 4A). For example, the greatest contractile forces
for 2M patches were generated with a pretension range of 500 to
2000 .mu.N (FIG. 4E). Based on this preliminary pretension, the
high and the low rate contractile forces of 1M to 6M patches were
then recorded and averaged. One of the 4M patches spontaneously
generated the greatest high rate contractile force (2141 .mu.N) and
the greatest low rate contractile force (2483 .mu.N) without any
treatment. The real muscle tissue layers of 4M and 6M patches were
thicker than 2M (FIG. 7P); however, the signal volume indexes of
collagen type I within the three densities were not significantly
different. This suggests that the myofibril content in 4M to 6M
patches may be higher than that in 2M patches. As such, the
difference in myofibril content may explain the greater twitch
forces generated from the 4 to 6M patches than the 2M. To determine
the underlying mechanisms for arrhythmic contractions exhibited in
4M to 6M patches, the signal volume index of the gap junction
protein Cx43 was examined. A statistical difference of Cx43 signal
volume indexes in 4M and 6M patches was observed when compared with
the 2M patches (p<0.05 or p<0.01) (FIG. 7Q). Cx43 is the
major protein of cardiac ventricular gap junctions and is crucial
to cell-cell communication and cardiac function. Recent works
reported that changed expression of Cx43 might contribute to higher
level of arrythmogenicity.
[0043] In some embodiments, the present disclosure provides a
method for fabricating a three-dimensional artificial cardiac patch
construct. Such a method is illustrated in FIG. 1 and may include
one or more of the following steps: coating a substrate with an
organic polymer; allowing the organic polymer coated on the
substrate to air dry; mounting anchors on the organic polymer
coating; sterilizing the coating of the organic polymer; forming a
biodegradable gel-based support scaffold on top of the organic
polymer; seeding neonatal cardiac cells on the biodegradable
gel-based support scaffold; culturing the neonatal cardiac cells in
vitro to form the three-dimensional artificial cardiac patch
construct. In general the culture conditions used may be suitable
for allowing the cells to self-organize into a real cardiac layer
and detach from the substrate to form the three-dimensional cardiac
patch. In some embodiments of the present disclosure, the organic
polymer is a silicone elastomer. In some embodiments, the silicone
elastomer is polydimethylsiloxane elastomer (PDMS). In some
embodiments of the present disclosure, the biodegradable gel-based
support scaffold is biocompatible and non-immunogenic. In some
embodiments, the biodegradable gel-based support scaffold is
fibrin. In some embodiments of the present disclosure, the seeding
of the biodegradable gel-based support scaffold comprises layering
the neonatal cardiac cells onto the scaffold. In some embodiments,
the seeding of the biodegradable gel-based support scaffold
comprises embedding the neonatal cardiac cells into the
biodegradable gel-based support scaffold. In some embodiments, the
neonatal cardiac cells are diluted in culture media prior to
seeding on the biodegradable gel-based support scaffold. In some
embodiments of the present disclosure, the real cardiac layer
comprises the neonatal cardiac cells and naturally produced
extracellular matrix on top of the biological support scaffold. In
some embodiments, the thickness of the real cardiac layer is
dependent on the density of the neonatal cardiac cells initially
seeded. In some embodiments of the present disclosure, the
fabricated artificial three-dimensional patch is spontaneously
contractile. In some embodiments of the present disclosure, the
artificial three-dimensional patch exhibits angiogenic bud
formation. In some embodiments, the artificial three-dimensional
patch exhibits active cell proliferation.
[0044] Further embodiments of the present disclosure pertain to a
three-dimensional artificial cardiac patch made by the methods of
the present disclosure. Additional embodiments of the present
disclosure relate to a method of treatment of cardiac tissue injury
in a subject in need thereof utilizing the three-dimensional
artificial cardiac patch construct disclosed herein.
[0045] As set forth in detail herein, the methods and compositions
of the present disclosure have numerous embodiments and variations.
In particular, various types of organic polymers may be used to
coat the substrate. Likewise, various types of biodegradable
gel-based support scaffolds may be formed on top of the organic
polymer coating. In addition, various types of cardiac cells may be
seeded in the support scaffold. Furthermore, the density of the
cardiac cells seeded may be varied to modulate the thickness of the
patch formed, the rate as well as the force of contraction.
[0046] Organic Polymers
[0047] In some embodiments of the present disclosure, the organic
polymer may be a silicone elastomer. In a related embodiment the
silicone elastomer may be polydimethylsiloxane elastomer (PDMS). In
some embodiments, anchors may be mounted on the organic polymer
coating and secured to the substrate. Suitable anchors that may be
used include minutien pins.
[0048] Cells
[0049] In a preferred embodiment, the cell types that may be used
to generate the three-dimensional artificial cardiac patch
construct of the present disclosure may include, but are not
limited to, cardiomyocytes, endocardial cells, cardiac adrenergic
cells, cardiac fibroblasts, vascular endothelial cells, smooth
muscle cells, stem cells, cardiac progenitor cells, and myocardial
precursor cells. Depending on the application of the
three-dimensional artificial cardiac patch and the type of cardiac
tissue material that is desired, the above types of cells may be
used independently or combined with one another. In one embodiment,
the three-dimensional artificial cardiac patches may be composed of
primary tissue isolates from the heart. Accordingly, small samples
of autologous, allogenic or xenogeneic donor cells may be used for
fabricating the three-dimensional artificial cardiac patch
construct. Alternatively, cells such as non-immunogenic universal
donor cell lines or stem cells may be used so long as they can be
manipulated to form the three-dimensional artificial cardiac patch
construct.
[0050] In certain embodiments, stem cells useful for the
compositions and methods provided herein include, for example,
embryonic stem cells, amniotic stem cells, bone marrow stem cells,
placenta-derived stem cells, embryonic germ cells, cardiac stem
cells, CDCs, induced pluripotent stem cells, mesenchymal stem
cells, endothelial progenitor cells, and spermatocytes. The stem
cells employed can be autologous or heterologous to the subject
being treated. In specific embodiments, the stem cells are
autologous stem cells.
[0051] The stem cells can be obtained or derived from any of a
variety of sources. For example, subjects that can be the donors
(or recipients) of stem cells in the methods and compositions
presented herein include, for example, mammals, such as
non-primates (e.g., cows, pigs, horses, cats, dogs, rats or
rabbits) or primates (e.g., monkeys or humans). In specific
embodiments, the subject is a human. In one embodiment, the subject
is a mammal, e.g., a human, such as a human with a congenital heart
defect or acute or chronic heart failure or other cardiac tissue
injury.
[0052] In a preferred embodiment of the present disclosure, freshly
isolated neonatal cardiac cells may consist of fibroblasts,
cardiomyocytes, smooth muscle cells, endothelial cells and cardiac
stem cells. The proliferation rate of cardiac cells is higher in
the fetal stage than that in the neonatal stage, and greatly
diminishes in adulthood. In an embodiment of the present
disclosure, 2-3-day-old rat pup heart cells may be used to
construct the three-dimensional artificial cardiac patch disclosed
herein.
[0053] Biodegradable Gel-Based Support Scaffolds
[0054] Biodegradable gel-based support scaffolds that may be used
to generate the three-dimensional artificial cardiac construct of
the present disclosure may include, but are not limited to
collagen, alginate, chitosan, fibrin, fibronectin, matrigel, small
intestine submucosa, and acellular tissue.
[0055] In some embodiments of the present disclosure the
biodegradable gel-based support scaffold may be biocompatible or
non-immunogenic. In a preferred embodiment the biodegradable
gel-based support scaffold may be fibrin. Fibrin is a natural
self-assembling biopolymer with many interesting properties, such
as biocompatibility, bioresorbability, ease of processing, ability
to be tailored to modify the conditions of polymerization, and
potential for incorporation of both cells and cell mediators.
Fibrin is used by the body to form clots along damaged endothelium.
Fibrin gels possess high seeding efficiency, uniform cell
distribution, and adhesion capabilities. Fibrin alone, or in
combination with other materials, has been used as a biological
scaffold for stem and primary cells to regenerate adipose tissue,
bone, cardiac tissue, cartilage, liver, nervous tissue, ocular
tissue, skin, tendons, and ligaments.
[0056] In an embodiment, the fibrin is formed by mixing thrombin
and fibrinogen solutions. In an embodiment of the present
disclosure the biodegradable gel-based support scaffold is also
non-immunogenic. Thrombin and fibrinogen which react to form fibrin
gel can be produced from the recipient's own blood, thus no the
potential risk of foreign body reaction or infection will occur
when used as a component in clinical application. In an embodiment
of the present disclosure, the fibrin support scaffold may be
developed with human fibrinogen and thrombin and its physical
properties may be characterized by histology and
immunohistochemistry.
[0057] In some embodiments of the present disclosure the neonatal
cardiac cells may be layered on to the biodegradable gel-based
support scaffold. In an alternative embodiment, the neonatal
cardiac cells may be embedded into the biodegradable gel-based
support scaffold.
[0058] In a related embodiment the neonatal cardiac cells may be
diluted in culture media prior to seeding on the biodegradable
gel-based support scaffold.
[0059] In an exemplary embodiment, the width of cardiomyocytes from
adult rats aged from 8 to 24 weeks was quantified to be 26.1-30.6
.mu.m, and the length as 123.3-148.8 .mu.m. In an embodiment of the
present disclosure, the pore sizes of the fibrin support scaffold
in the three-dimensional artificial cardiac patch, were 15.0-150.0
.mu.m (FIGS. 6A and 6B), which is a suitable compartment for a
cardiomyocyte that may facilitate cell organization and
interconnectivity.
[0060] In some embodiments the present disclosure provides for the
formation of a real cardiac layer on top of the biological support
scaffold. In a related embodiment, the real cardiac layer may
include the neonatal cardiac cells and naturally produced
extracellular matrix on top of the biological support scaffold.
Such a layer of cells is illustrated in FIGS. 7A-7C. In a preferred
embodiment, the thickness of the real cardiac layer formed may be
modulated by varying the density of the cells layered or embedded
in the biodegradable gel-based support scaffold. The reason for
this difference may result from the lower cell density and/or the
different growth rate of each cell type within the patch. In an
exemplary embodiment, the optimal cell density, using rat neonatal
cardiac cells, for the formation of the three-dimensional
artificial cardiac patch may be 4 million (4M).
[0061] In an embodiment of the present disclosure the
three-dimensional artificial cardiac patch is spontaneously
contractile. In a preferred embodiment, the rate of spontaneous
contraction of the cardiac patch may be dependent on the density of
the neonatal cardiac cells initially seeded. In a related
embodiment, the three-dimensional artificial cardiac patch may
exhibit contractile twitch force. In a preferred embodiment the
contractile twitch force may me modulated by varying the density of
the neonatal cardiac cells initially seeded. In some embodiments of
the present disclosure, the three-dimensional artificial patch may
exhibit angiogenic bud formation. Further, in an embodiment of the
present disclosure, vascularity of the three-dimensional artificial
cardiac patches may be determined by staining for various
endothelial cell markers to show presence of angiogenesis buds
capable of facilitating media perfusion. The microvasculature
within the patch may be suitable to supply blood and nutrients into
the patch during in vivo applications.
[0062] In related embodiments of the present disclosure, the
three-dimensional artificial cardiac patch may exhibit active cell
proliferation. The viability of freshly isolated heart cells may be
determined by using established isolation protocols. Furthermore,
in another embodiment of the present disclosure, the proliferation
of the cells within the three-dimensional artificial cardiac patch
may be assessed by staining for somatic cell proliferation markers.
In an embodiment, the somatic cell proliferation marker stained for
is the Ki67 nuclear and nucleolar protein.
[0063] In some embodiments the present disclosure provides for
implanting the fabricated three-dimensional artificial cardiac
construct in a suitable recipient. In a related embodiment, the
recipient may be suffering from a congenital heart disease. In an
embodiment the congenital heart disease is selected from a group
consisting of Hypoplastic left heart syndrome, tetralogy of fallot,
ventricular septal defects, atrial septal defects, endocardial
cushion defect. In another embodiment, the recipient may be
suffering from a cardiac tissue injury. In a preferred embodiment,
the cardiac tissue injury may be caused by acute or chronic stress,
atheromatous disorders of blood vessels, ischemia, myocardial
infarction, inflammatory disease and cardiomyopathies or
myocarditis. In some embodiments, the present disclosure provides a
method for the treatment of a cardiac tissue injury. Such a method
may include implanting the three-dimensional cardiac construct
disclosed herein in a subject in need thereof. In a related
embodiment the cardiac tissue injury may be due to acute or chronic
stress, atheromatous disorders of blood vessels, ischemia,
myocardial infarction, inflammatory disease and cardiomyopathies or
myocarditis. In some embodiments, the acute or chronic stress may
be due to systemic hypertension, pulmonary hypertension or valve
dysfunction. In some embodiments of the present disclosure, the
atheromatous disorder of blood vessels is coronary artery disease.
In another embodiment of the present disclosure, there is provided
a composition comprising the three-dimensional artificial cardiac
patch disclosed herein. In an embodiment the present disclosure
pertains to a three-dimensional artificial cardiac patch comprising
an organic polymer coated on a substrate; a biodegradable gel-based
support scaffold formed on top of the organic polymer; and cardiac
cells, where the cardiac cells are seeded on the biodegradable
gel-based scaffold. In an embodiment, the three-dimensional
artificial cardiac patch further comprises at least one agent from
the group consisting of survival factors, growth factors,
pharmacological agents, angiogenic factors, beta-blockers or ACE
inhibitors.
[0064] In a related embodiment there is also provided a medicament.
Such a medicament includes the three-dimensional artificial cardiac
patch disclosed herein. In a related embodiment, the medicament of
the present disclosure further comprises at least one agent from
the group consisting of survival factors, growth factors,
pharmacological agents, angiogenic factors, beta-blockers or ACE
inhibitors.
[0065] In an exemplary embodiment of the present disclosure, the
three-dimensional artificial cardiac patch may be fabricated using
2-3-day-old rat pup heart cells on a fibrin gel-based support
scaffold. In a related embodiment, modulation of the contraction
rate of the three-dimensional artificial cardiac patch by varying
the density of the cells embedded in or layered on the fibrin gel
was observed. The spontaneous contractions in the three-dimensional
artificial cardiac patches were recorded and measured at day 2 from
the cell densities of 2 million (2M) to 6 million (6M). Earlier
tissue contractions were observed for a few of the patches with
cell densities of 4 million (4M) to 6 million (6M) after only one
day of incubation. The contraction rate at day 2 increased with
higher cell densities. The average contraction rate for a 6M patch
was 430.+-.54 bpm (n=23) at day 2, which falls within the range of
a normal adult rat heartbeat. For 1 to 3M densities, the highest
mean contraction was at day 4, and then steadily decreased from day
6 to 8. For 4 to 6M densities, the highest contraction occurred
much earlier, at day 2, but it decreased sharply from day 4 to 8
(FIG. 3).
[0066] In the exemplary embodiment, the real cardiac layers formed
for 4M and 6M patches were thicker than 2M (FIG. 7N). The same
signal volume indexes of collagen type I within the three densities
suggest that the myofibril content in 4 to 6M patches was higher
than that in 2M patches. As such, the difference in myofibril
content may explain the greater twitch forces generated from the 4
to 6M patches than the 2M. To determine the underlying mechanisms
for arrhythmic contractions exhibited in 4 to 6M patches,
Applicants examined the signal volume index of the gap junction
protein Cx43. Cx43 is the major protein of cardiac ventricular gap
junctions and is crucial to cell-cell communication and cardiac
function. Recent works reported that reduced expression and
enhanced lateralization of Cx43 might contribute to enhanced
arrythmogenicity. In an exemplary embodiment, no statistical
difference of Cx43 signal volume index in 4M and 6M patches was
observed as compared to 2M ones, yet, there was a slight trend
showing that the expression of Cx43 was relatively higher in 2M
patches (FIG. 7P).
[0067] In an exemplary embodiment, the amplitude of contractile
twitch force generated and the onset and rate of contractions was
modulated with cell densities. The patch with 4 million cells
generated the greatest high and low rate contractile twitch forces,
and the contraction rate of a patch with 6 million cells resembled
an adult rat heart rate, which, as of yet, were the best reported.
In addition, patches manifested flourishing angiogenesis and
cellular division. Further modulation is needed to enable media
perfusion throughout the entire patch, and will result in a more
robust 3D artificial cardiac patch
Advantages
[0068] The methods of the present disclosure may be utilized to
make three-dimensional artificial cardiac patches for various
applications. For instance the methods of the present disclosure
may be used for repairing cardiac tissue injuries and congenital
heart defects. The methods of the present disclosure may also be
used for the development of biocompatible, adaptive,
non-immunogenic materials for cardiac tissue replacement. The
three-dimensional artificial cardiac patches of the present
disclosure revealed better contractility than ever reported before
for engineered cardiac tissue. Additionally, the cardiac patches of
the present disclosure display abundant vascularization and robust
cellular division. Furthermore, in some embodiments of the present
disclosure, the patches may be constructed using host origin
fibrinogen and thrombin to produce the non-immunogenic fibrin
scaffold before in vivo application.
[0069] Reference will now be made to various embodiments of the
present disclosure and experimental results that provide support
for such embodiments. However, Applicants note that the disclosure
herein is for illustrative purposes only and is not intended to
limit the scope of the claimed subject matter in any way.
Example 1
Isolation of Primary Cardiac Cells
[0070] Cardiac cells were isolated from the hearts of 2-3 day old
neonatal Sprague-Dawley rats using an established method. Briefly,
each heart was cut into 3-4 pieces in an ice-cold phosphate buffer
consisting of 116 mM NaCl, 20 mM HEPES, 1 mM Na.sub.2HPO.sub.4, 5.5
mM glucose, 5.4 mM KCl and 0.8 mM MgSO.sub.4. After blood cells
were rinsed out, heart pieces were transferred to a dissociation
solution (DS) consisting of 0.32 mg/ml collagenase type 2-filtered
(Worthington Biochemical Corporation, Lakewood, N.J.) and 0.6 mg/ml
pancreatin in phosphate buffer. The hearts were cut into 1 mm.sup.2
pieces and then transferred to an orbital shaker and maintained at
37.degree. C. for 30 minutes at 60 rpm. At the end of the digestion
process, the supernatant was collected in 3 ml of horse serum to
neutralize the enzyme and centrifuged at 1000 rpm for 5 minutes at
4.degree. C. The cell pellet was resuspended in 5 ml horse serum
and kept in an incubator at 37.degree. C. supplied with 5%
CO.sub.2. Fresh DS was added to the partially-digested tissue and
the digestion process was repeated an additional 2-3 times. Cells
from all the digests were pooled, centrifuged and suspended in
culture medium (CM) consisting of 320 ml M199 (Life Technologies,
Grand Island, N.Y.), with 20% F12k (Life Technologies, Grand
Island, N.Y.), 10% fetal bovine serum, 5% horse serum, 1%
antibiotic-antimycotic, 40 ng/ml hydrocortisone and insulin 100
ng/ml. Cell viability was analyzed by Trypan blue solution (4%)
staining according to the manufacturer's protocol and the
percentage of live cells determined.
Example 2
Fabrication of Artificial Cardiac Patch
[0071] The method to fabricate the cardiac patch is shown in FIGS.
1A-1H. Briefly, a 35 mm tissue culture plate was coated with 2 ml
of SYLGARD (PDMS, type 184 silicone elastomer) (Dow Chemical
Corporation, Midland, Mich.). The plate was air dried for 2 weeks
and sterilized with 80% ethanol before use. Four minutien pins
(Fine Science Tools, Foster City, Calif.), 0.1 mm diameter, were
placed in the culture plate to form a 2 cm.times.2 cm square. The
fibrin gel was made by plating 1 ml of CM containing 10 U/ml
thrombin and adding 500 .mu.l of saline containing 20 mg/ml
fibrinogen, and well mixed to promote the formation of gel within
15 minutes. Primary cardiac cells were diluted in CM at a pre-set
density and 2 ml of the cell suspension CM was transferred to the
culture plate. Aminocaproic acid (2 mg/ml) was added to the culture
plate to inhibit the fibrinolysis by endogenous proteases. The
cells were cultured in an incubator at 37.degree. C. and 5%
CO.sub.2 with CM changes every other day.
Example 3
Patch Formation and Contraction Rate
[0072] Two days after cell plating, cultured cardiac constructs
began contraction and fibrin gels detach from the rim of culture
plates. At days 2, 4, 6, and 8, the patch growth progress was
captured in still photographs and videos using a camera (Lumena,
Ottawa, ON) mounted on an inverted phase-contrast microscope
(Olympus, Center Valley, Pa.). The movies were slowly replayed and
the contraction rates manually counted (FIG. 8 showing Movie
1).
Example 4
Contractile Twitch Force and Electrocardiogram (ECG)
[0073] From day 4, twitch force was measured using a high
sensitivity isometric force transducer (MLT0202, ADinstruments,
Colorado Springs, Colo.), connected to a quad bridge amplifier
(FE224, ADinstruments, Colorado Springs, Colo.),
electrophysiological signal was measured using Octal Bio Amp
(ML138, ADinstruments, Colorado Springs, Colo.). Data was acquired
through a 16 channel PowerLab system (PL3516/P, ADInstruments,
Colorado Springs, Colo.). As shown in FIG. 1J, the contractile
force was measured by attaching the force transducer arm to one
free-corner of the square patch, while the other three ends were
held fixed. In order to obtain the Frank-Starling relationship of
contractile force, pretension was adjusted using a
micro-manipulator (Radnoti LLC, Monrovia Calif.) and measurements
of spontaneous contraction were recorded. Electrocardiogram (ECG)
of the patch was measured by inserting the cathode into the center
of the patch and the anode in one of the four patch corners. The
media immersing the patch was used as ground. Spontaneous
contractile force and ECG measurements were recorded for 30-60
seconds. LabChart (ADInstruments, Colorado Springs, Colo.) was used
for data analysis. The peak analysis module was used to calculate
the maximum twitch force and baseline force (pretension). The ECG
analysis module was used to calculate the R wave amplitude.
Example 5
Morphology
[0074] Seven days after plating, formed patches were trimmed, and
from the central part of the patch two 0.5.times.0.5 cm blocks were
taken, placed in a peel-a-way disposable embedding mold (VWR
International, Radnor, Pa.) and frozen in liquid nitrogen, and then
immediately, immerged in Tissue Tek OCT compound (VWR
International, Radnor, Pa.), and immediately placed in a
-80.degree. C. freezer. Once the OCT compound was completely solid,
each sample was sliced using a cryotome (Thermo Fisher Scientific,
Waltham, Mass.). Tissue cross- and planar-sections were cut at a
thickness of 10 .mu.m or 6 .mu.m. The sections were placed on
VWR.RTM. Microslides for preparation of morphological and
immunofluorescence examinations. For measurement of physical
properties such as fibrin scaffold thickness and pore size, images
from both cross- and planar-sections of 6 .mu.m thickness were
taken directly under a light microscope (Olympus, Center Valley,
Pa.) and fibrin scaffold thickness was calculated with ImageJ 1.47d
(Wayne Rashand, National Institute of Health, USA). For measurement
of the real heart tissue (a layer of cells and naturally produced
extracellular matrix forms on top of the fibrin gel scaffold)
thickness on the fibrin scaffold, cross- and planar-sections of 10
.mu.m thickness were stained with Masson's trichrome reagents
according to manufacturer's protocol and images were taken under a
light microscope. The distinct tissue layers were traced and
thicknesses calculated.
Example 6
Immunofluorescence
[0075] For measurement of the signal volumes of connexin 43 and
collagen type I, 6 .mu.m thickness cross-sections were fixed in ice
cold acetone for 10 minutes, nonspecific epitope antigens were
blocked with 10% goat serum at room temperature for 1 hour.
Sections were incubated with specific mouse anti-.alpha.-actinin
monoclonal antibody (Sigma, Catalog No A7811) 1:200, rabbit
anti-connexin 43 (Cx43) (Abcam, ab11370) 1:100, rabbit anti-von
Willebrand factor (vWF) (Abcam, ab6994) 1:750, rabbit anti-ki 67
(Abcam, ab66155) 1:100, rabbit anti-collagen type I (Abcam,
ab34710) 1:100 at room temperature for 1 hour, and treated with
goat anti-mouse and goat anti-rabbit secondary antibodies (Alexa
Fluor 488, Alexa Fluor 546, and Alexa Fluor 633, Life Technology)
1:400 at room temperature for 1 hour. Nuclei were counterstained
with DAPI (2.5 .mu.g/ml) for 5 min at room temperature. For
observation of endothelial cell growth and nuclear division in the
patch, a modified immunostaining protocol of tissue constructs was
used. Fresh tissue patches were directly fixed in ice cold acetone
for 10 minutes. 1.0.times.1.0 tissue patch blocks from the central
part of the patches were trimmed and nonspecific epitope antigens
were blocked and cell membranes permeated with 10% goat serum per
0.5% Triton X-100 at room temperature for 45 minutes. Tissue patch
blocks were then incubated in mouse anti-a-actin antibody 1:200,
rabbit anti-von Willebrand factor (vWF) (Abcam, ab6994) 1:750,
rabbit anti-ki67 (Abcam, ab66155) 1:100, and rabbit anti-Cx43 1:100
at room temperature for 2 hours. The rest of the steps of
immunostaining for tissue patches were same as that for
cross-sections. Fluorescent images were obtained with a Nikon
C2.sup.+ confocal laser scanning microscope (Nikon Instruments Inc.
Melville, N.Y.). For measurement of the changes of gap junctions,
collagens and myofibrils, signal volumes of Cx43, collagen type I
and .alpha.-actinin were examined within cross-sections. Two movies
from each sample were acquired with a signal depth of 8 .mu.m
scanned by 33 frames. After determining specific thresholds for
Cx43, collagen type I and .alpha.-actinin, signal volume and
intensity for each sample were measured. The relative changes of
Cx43 (or collagen type I) for different cell densities were
expressed with
Cx43Index=(Cx43volume*intensity)/(.alpha.-actinin
volume*intensity)
and averaged for each sample.
Example 7
Statistics
[0076] Results are presented as mean.+-.standard deviation.
Chi-Square analysis was used to test frequency variables.
Comparisons among the three groups were made with a one-way
analysis of variance (ANOVA), followed by the Bonferroni post hoc
comparison test; in addition, Kruskal-Wallis test were performed.
In all tests, differences were considered statistically significant
at a value of p<0.05.
Example 8
Patch Formation
[0077] By the present established isolation method, cell viability
was 81.0.+-.2.2% (n=16). The time required for patch formation was
a function of the initial plating density. Representative
progression of patch formation of 1, 3 and 5 million cells (M) per
dish is shown in FIG. 2. At day 4, formation was complete for 28.0%
(7/25), 56.3% (9/16), and 40.0% (6/15) of patches formed with 1, 3
and 5M densities, respectively; Pearson Chi-Square analysis
indicated p=0.195. At day 6, there were 68.0% (17/25), 87.5%
(14/16), and 60.0% (9/15) of patches formed with 1, 3 and 5M
densities, respectively; Fisher's Exact test demonstrated p=0.232.
The results indicated that though there were no statistical
differences among the plating densities, patches with 3M density
exhibited the highest percentage of patch formation both at days 4
and 6. From day 4 the tissue detachments were seen in the 5M and 6M
density patches (arrow in FIG. 2).
Example 9
Contraction Rate
[0078] From day 2 the spontaneous tissue contractions were seen
under microscope from the dishes with 2M-6M densities. The average
contraction rates for 2, 3, 4, 5 and 6M were 115.+-.56 (n=27),
124.+-.42 (n=16), 207.+-.107 (n=23), 279.+-.174 (n=8) and 430.+-.54
bpm (n=23) respectively; Kruskal-Wallis test showed significant
differences (p<0.01) with mean ranks 27.8, 32.7, 48.7, 57.0 and
82.7 respectively. At day 4 the average contraction rates for 1, 2,
3, 4, 5 and 6M were 169.+-.54 (n=25), 222.+-.74 (n=37), 207.+-.38
(n=16), 48.+-.23 (n=17), 35.+-.31 (n=12) and 57.+-.59 bpm (n=14)
respectively; Kruskal-Wallis test analyzed significant difference
(p<0.01) with mean ranks 69.2, 87.3, 87.0, 26.5, 18.3 and 25.7
respectively. Then the average contraction rates for each cell
density decreased at days 6 and 8 (FIG. 3).
Example 10
Contractile Twitch Force
[0079] High rate (65-270 bpm) and low rate (<20 bpm) twitch
forces were recorded from formed patches from day 4 to 6. For 1, 2
and 3M patches, the high rate rhythmic contractions were detected
throughout the entire recording period (starting at the onset of
pretension); the largest twitch force was recorded when the
pretension was set between 500 to 2000 .mu.N (FIGS. 4A, 4B).
However, for 4, 5 and 6M patches, high rate contractions were only
detected within the a few seconds after a pretension load was
applied, after which low rate contractions were observed (FIG. 4C,
4D). FIG. 4E illustrates the effects of pretension on the magnitude
of contractile force for 2M density patches. For, the pretension
ranges of 3000-2500, 2500-2000, 2000-1500 1500-1000, 1000-500 and
500-0 .mu.N, the contractile forces were 377.+-.154 .mu.N (n=4),
445.+-.213 .mu.N (n=8), 583.+-.238 .mu.N (n=8), 621.+-.200 .mu.N
(n=8), 584.+-.195 .mu.N (n=8) and 409.+-.126 .mu.N (n=8),
respectively (Bonferroni post hoc p>0.05 for all).
[0080] Representative samples of the greatest high and low rate
contractile forces recorded by a pretension range of 1000 to 2000
.mu.N from 2, 4 and 6M densities are shown in FIG. 4B, FIG. 4C and
FIG. 4D. The maximum high rate contractile forces were 932, 2160
and 2141 .mu.N and the maximum low rate contractile forces were
1044, 2483 and 2364 .mu.N for 2, 4 and 6M, respectively. The
average high rate contractile forces for 1 through 6M patches
loaded with a pretension range of 1000 to 2000 .mu.N showed a
significant difference (Kruskal-Wallis p<0.01). The 1-3M patches
had an average contractile force in the 300-500 .mu.N ranges,
whereas the 4-6M patches possessed a contractile force in the
1300-1700 .mu.N ranges, with the 4M patch showing the highest
contractile force (FIG. 4F). The average low rate twitch forces for
1 to 6M patches loads with pretension between 1000 and 2000 .mu.N
also showed a significant difference (Kruskal-Wallis p<0.01).
The 1-3M patches had an average contractile force in the 1500-2000
.mu.N ranges, with the 4M patch showing the highest contractile
force (FIG. 4G).
[0081] Electrocardiogram
[0082] The representative ECG graphs for 2, 4 and 6M patches with
high rate contraction are shown in FIGS. 5A-5C. FIG. 5D is the
synchronized contraction graph of the 6M patch. The QRS complex
pattern from 2 and 4M patches closely resembles that of an adult
rat heart. The R-wave amplitudes from 4M (0.0244.+-.0.0087, n=10)
and 6M (0.0281.+-.0.0064 mV, n=5) patches were greater than that
from 2M (0.0089.+-.0.0081 Mv, N=8) patch (Bonferroni post hoc
p<0.001).
Example 11
Morphology
[0083] The physical properties of fibrin gel scaffolds of formed
patches were examined at day 7 (FIG. 6). A layer of cardiac cells
and self-produced extracellular matrix proteins, which comprises
the real cardiac layer, formed on top of the fibrin gel scaffold
(FIG. 6A). The total cross-sectional depth of the patches was
700-1000 .mu.M (FIG. 6A) and the planar networks of the fibrin gel
scaffolds are depicted in FIGS. 6B and 6C. The thickness (FIG. 6D)
and planar texture (FIGS. 6E and 6F) of the real cardiac layer were
further revealed by Masson trichrome staining though they were
physically altered by fixing process. The planar section shows that
the real cardiac tissue is closely incorporated into the lower
fibrin network (FIG. 6E). The positive staining for the ki67
(white) (FIGS. 6G and 6H) suggests the presence of robust nuclear
division, the positive staining for vWF (red) (FIGS. 6G and 6I)
suggests that endothelial cells were proliferating, and the
positive staining for connexin 43 (yellow) (FIG. 6I) indicates
intracellular coupling via gap junctions in the artificial
tissue.
[0084] Samples stained with Masson's trichrome were used to
illustrate the thickness of muscle tissue and support fibrin
scaffold, as shown in FIGS. 7A, 7B and 7C from patches with 2, 4
and 6M densities. Cross-sections in FIGS. 7D-7O show the
expressions of myofibrils by .alpha.-actinin staining and gap
junctions by Cx43 staining for 2, 4 and 6M. The average thicknesses
of real muscle layers were 18.2.+-.2.4 (n=13), 21.4.+-.1.4 (n=14)
and 20.6.+-.2.4 (n=12), respectively; there were significant
differences when comparing 4M and 6M (Bonferroni post hoc p<0.05
or p<0.01) with 2M (FIG. 7P). The signal volume index of Cx43
was greater for 2M (0.182.+-.0.051, n=13), than for 4M
(0.132.+-.0.039, n=13) and 6M (0.126.+-.0.038, n=16) (Bonferroni
post hoc p<0.05 or p<0.01) (FIG. 7Q). The signal volume index
of collagen type I was also calculated to be (0.221.+-.0.065, n=9),
(0.209.+-.0.070, n=8) and (0.196.+-.0.050, n=15) for 2M, 4M and 6M,
respectively; however, there were no obvious trends or significant
statistical differences (Bonferroni post hoc p>0.05).
[0085] In summary, Applicants report the fabrication of a
three-dimensional artificial cardiac patch using neonatal heart
cells on a fibrin gel scaffold. The amplitude of contractile twitch
force generated and the onset and rate of contractions was
modulated with cell densities. The patch with 4 million cells
generated the greatest high and low rate contractile twitch forces,
and the contraction rate of a patch with 6 million cells resembled
an adult rat heart rate, which, as of yet, were the best reported.
In addition, patches manifested flourishing angiogenesis and
cellular division.
[0086] The embodiments described herein are to be construed as
illustrative and not as constraining the remainder of the
disclosure in any way. While the embodiments have been shown and
described, many variations and modifications thereof can be made by
one skilled in the art without departing from the spirit and
teachings of the invention. Accordingly, the scope of protection is
not limited by the description set out above, but is only limited
by the claims, including all equivalents of the subject matter of
the claims. The disclosures of all patents, patent applications and
publications cited herein are hereby incorporated herein by
reference, to the extent that they provide procedural or other
details consistent with and supplementary to those set forth
herein.
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