U.S. patent application number 11/534917 was filed with the patent office on 2007-01-18 for engineered biografts for repair of damaged myocardium.
This patent application is currently assigned to Ben-Gurion University of the Negev. Invention is credited to Smadar Cohen, Ayelet Dar, Sharon Etzion, Jonathan Leor, Anat Perets, Sigalit Shaprut.
Application Number | 20070014772 11/534917 |
Document ID | / |
Family ID | 37661865 |
Filed Date | 2007-01-18 |
United States Patent
Application |
20070014772 |
Kind Code |
A1 |
Cohen; Smadar ; et
al. |
January 18, 2007 |
ENGINEERED BIOGRAFTS FOR REPAIR OF DAMAGED MYOCARDIUM
Abstract
A method for repairing a damaged myocardium in a mammal,
comprising: a) providing a three-dimensional porous polysaccharide
matrix; b) introducing mammalian cells into said matrix; c) growing
said cells in said matrix in vitro, until a tissue-engineered
biograft is formed, comprising a contracting tissue; and d)
transplanting the tissue-engineered biograft onto the myocardial
tissue or myocardial scar tissue of said mammal, optionally
previously removing scar or dead tissue from the site of
implantation.
Inventors: |
Cohen; Smadar; (Beer Sheva,
IL) ; Dar; Ayelet; (Rehovot, IL) ; Etzion;
Sharon; (Beer Shevea, IL) ; Perets; Anat;
(Bet-Shemesh, IL) ; Shaprut; Sigalit; (Beer-Sheva,
IL) ; Leor; Jonathan; (Gane' Tikva, IL) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Ben-Gurion University of the
Negev
Beer Sheva
IL
|
Family ID: |
37661865 |
Appl. No.: |
11/534917 |
Filed: |
September 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09654276 |
Sep 1, 2000 |
|
|
|
11534917 |
Sep 25, 2006 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/366 |
Current CPC
Class: |
A61L 27/3873 20130101;
C12N 5/0657 20130101; C08L 5/04 20130101; A61L 27/56 20130101; A61L
27/3804 20130101; A61L 27/20 20130101; C12N 2501/165 20130101; A61L
27/3886 20130101; A61L 27/20 20130101; C12N 2533/74 20130101; C12N
5/0691 20130101 |
Class at
Publication: |
424/093.7 ;
435/366 |
International
Class: |
C12N 5/08 20060101
C12N005/08; A61K 35/34 20070101 A61K035/34 |
Claims
1-17. (canceled)
18. A tissue-engineered cardiac biograft for transplantation into
myocardial tissue or myocardial scar tissue, comprising: a porous
polysaccharide matrix comprising controlled-release polymeric
microspheres capable of releasing soluble angiogenic growth
factors, wherein said matrix is non-adhesive; and mammalian cells
comprising fetal, autologous, or allogeneic cardiomyocytes alone or
in combination with at least one of fibroblasts, smooth muscle
cells, or endothelial cells that are fetal, autologous, or
allogeneic; wherein said cells have been cultured in said matrix in
vitro and the cells form a multicellular aggregate.
19. A tissue-engineered cardiac biograft according to claim 18,
wherein said polysaccharide is an alginate.
20. A tissue engineered biograft according to claim 18, wherein
said cardiomyocytes are fetal cardiomyocytes, neonatal
cardiomyocytes, or adult cardiac cells.
21. A method of preparing a three-dimensional tissue-engineered
biograft comprising: a) providing a porous polysaccharide matrix
comprising microspheres capable of releasing soluble angiogenic
growth factors, wherein said matrix is non-adhesive; and b)
co-culturing the porous polysaccharide matrix in vitro with fetal,
autologous, or allogeneic mammalian cells comprising cardiomyocytes
alone or in combination with at least one of fibroblasts, smooth
muscle cells, or endothelial cells that are fetal, autologous, or
allogeneic, until a cardiac-like tissue is formed, comprising a
tissue-engineered biograft.
22. The method of claim 21, wherein the porous polysaccharide
matrix comprises an alginate polysaccharide.
23. The method of claim 21, wherein the porous polysaccharide
matrix generates a scaffold.
24. A method according to claim 21, wherein said cardiomyocytes are
fetal cardiomyocytes, neonatal cardiomyocytes, or adult cardiac
cells.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the use of
tissue-engineered cardiac biografts for transplantation into
damaged myocardium.
BACKGROUND OF THE INVENTION
[0002] Despite recent advances in the treatment of acute myocardial
infarction (MI), attempts to repair extensive myocardial damage and
to treat heart failure are often met with limited success. One of
the reasons for the lack of success is that the myocardium is
unable to regenerate because cardiomyocytes do not have the
capacity for replication after injury, and furthermore because
there are apparently no muscle stem cells in the myocardium.
[0003] Existing strategies for restoring heart function after
myocardial injury are practically limited to cardiac
transplantation. Since the supply of donor hearts is limited,
tissue engineering appears to be a promising approach for the
formation of new functional tissue to replace lost or failing
tissue. Earlier studies indicated the possibility of transplanting
isolated cardiomyocytes or myoblasts in order to enhance cardiac
function following myocardial injury (Taylor et al., Nat. Med.,
4:929-33, 1998). However, this approach does not permit the
formation of tissue-engineered cardiac biografts having the desired
shape, size and consistency.
[0004] A bioengineered cardiac graft using fetal myocardial cells
contained within a gelatin mesh was recently disclosed (Li et al.,
Circulation 19, 63-69, 1999). When transplanted into rodent
post-infarction myocardial tissue, however, no improvement in
cardiac function could be observed. A similar method for producing
a myocardial graft in a mammal using scaffolding is disclosed in
U.S. Pat. No. 6,099,832. In said patent, cardiomyocytes were either
directly introduced into a cryo-damaged myocardial tissue, or were
first suspended on scaffolding polymers prior to transplantation.
In both cases, the biograft did not provide tissue characteristics
such as cell-cell interactions and the formation of extracellular
matrix. In addition, the methods of U.S. Pat. No. 6,099,832 do not
provide the ability to determine the composition and consistency of
the biograft.
[0005] In WO 99/03973, mesenchymal stem cells were supported onto a
semi-solid or solid matrix, such as collagen and its derivatives,
polylactic acid or polyglycolic acid, and rapidly injected into the
tissue. However, cells were not implanted within a scaffolding-type
matrix prior to transplantation, and therefore there was no
creation of tissue-engineered cardiac biograft prior to
transplantation.
[0006] In WO 97/44070 of the same applicants hereof, the
specification of which is incorporated herein by reference, a new
method for the preparation of three-dimensional, porous,
biodegradable sponges made from polysaccharides is disclosed.
[0007] In WO 99/65463 of the same applicants hereof, the
specification of which is incorporated herein by reference, a
device for the delivery of drugs to mucosal or luminal surfaces
using a porous matrix is disclosed. According to a preferred
embodiment of said delivery device, the porous matrix comprises an
alginate scaffold. However, said porous matrix was used only for
mucosal drug delivery.
[0008] It has now been surprisingly found, and this is an object of
the present invention, that cultured mammalian cells may be grown
in vitro in porous three-dimensional alginate scaffolds, leading to
the formation of tissue-engineered biografts, said biografts
possessing the characteristics of myocardial tissue, including the
formation of cell-cell interactions, contractility and
extracellular matrix components. Said tissue-engineered biografts
may be used for repairing damaged myocardial tissue by
transplanting them onto said myocardial tissue.
[0009] It is a purpose of the present invention to provide a
tissue-engineered cardiac biograft system for use in replacing the
scar tissue that is formed following myocardial infarction.
[0010] It is a further object of the invention to provide a
tissue-engineered cardiac biograft system that is useful for
improving impaired cardiac functions following myocardial
infarction.
[0011] Other objects and advantages of the invention will become
apparent as the description proceeds.
SUMMARY OF THE INVENTION
[0012] The present invention, as opposed to procedures in which
cells are transplanted, enables a control of the tissue formation
process prior to transplantation. It is possible to control and
determine the shape and size of the graft, the consistency and
composition of the graft (e.g. the total number of cells and cell
to cell ratio in co-cultures), and its function prior to
transplantation. In addition, the scaffold of the present invention
is composed of polysaccharides and has a sponge-like morphology as
opposed to previous scaffolds which are based on gelatin and are in
a configuration of a mesh.
[0013] As said, the invention is primarily directed to the use of
3-D porous polysaccharide scaffold in the preparation of
tissue-engineered biograft for the transplantation of mammalian
cells into the heart, for the purposes of repairing damaged
myocardium. Polysaccharide scaffolds useful in the present
invention maybe, e.g., those described in WO 97/44070.
[0014] Preferably, tissue-engineered cardiac biografts is
constructed from mammalian cells that are selected from the group
consisting of fetal cardiac cells, neonatal cardiac cells,
fibroblasts, smooth muscle cells, endothelial cells, skeletal
myoblasts, mesenchymal stem cells and embryonic stem cells.
[0015] In another aspect, the invention is directed to a
tissue-engineered biograft for transplantation into myocardial
tissue or myocardial scar tissue, comprising a porous alginate
matrix containing mammalian cells, wherein said cells are cultured
in said matrix in vitro prior to transplantation.
[0016] The invention further provides a method for repairing a
damaged myocardium in a mammal in need of such repair, said method
comprising the steps of: [0017] a) providing a three-dimensional
porous polysaccharide matrix; [0018] b) introducing mammalian cells
into said matrix; [0019] c) growing said cells in said matrix in
vitro, until a tissue-engineered biograft is formed, comprising a
contracting tissue; and [0020] d) transplanting the
tissue-engineered biograft into the myocardial tissue or myocardial
scar tissue of said mammal, optionally removing scar or dead tissue
from the site of implantation.
[0021] In a preferred embodiment of the present invention, a porous
alginate matrix is used as a polysaccharide scaffolds.
[0022] The tissue-engineered biografts of the present invention
present a number of novel features, some of which will be
illustrated in the following description and Examples. One such
relevant feature is the formation of a contracting cardiac-like
tissue, characterized by cell-cell interactions and extracellular
matrix components within the tissue-engineered biograft prior to
transplantation. Another important feature is the extensive
neovascularization that is observed following implantation of the
bioengineered cardiac tissue into the infarcted myocardium. It
appears that the high degree of neovascularization contributes to
the prolonged survival of the cells in the grafts. A further
structural characteristic of the tissue-engineered biograft
technique is the integration of the tissue with the surrounding
myocardial tissue following transplantation. This integration is
seen both in terms of the formation of intercellular junctions and
tissue ingrowth from the host into the biograft. Furthermore, when
the cells used are fetal cardiomyocytes, it is possible to observe
differentiation of said fetal cells into mature cardiomyocytes,
together with their organization into bundles of myofibers. As a
result of all these integration processes, remodeling of the
damaged myocardium can take place. Such re-modeling, in turn,
causes an improvement in cardiac function, as witnessed by the
attenuation of ventricular dilation and the cessation of further
deterioration in contractility and critical pressure/flow
parameters that would otherwise occur as part of the normal
response to the myocardial lesion.
[0023] Many of the advantageous features of the tissue-engineered
biograft of the present invention are attributable to the use of
polysaccharides such as alginate matrices as scaffolds for the
seeded cells. The scaffolds are hydrophilic in nature, which
permits their rapid wetting by aqueous media and efficient cell
seeding. In addition, as a consequence of the large pore diameter
(between 100 and 150 .mu.m), the physical obstruction of liquid
flow by the tortuous pore pathway is minimal, allowing relatively
free movement of cells into the scaffold pores, with their
subsequent uniform distribution throughout the scaffold volume.
These matrices display a highly-porous structure, characterized by
a high degree of pore interconnectivity. The connectivity of the
pores in the alginate scaffolds allows the re-organization of the
dispersed cardiac cells into multicellular aggregates, with 3-D
cell-cell interactions. Furthermore, ECM components that are
secreted by the cells contribute to the compaction of the cell
aggregates into a tissue-like form. The contractility of the
mutlicellular aggregates indicates that the newly-organized tissue
maintains the characteristics of a cardiac tissue.
[0024] The ECM components were apparently synthesized by
cardiofibroblasts, which constitute, according to cell purity
assay, 30% of the seeded cells. It is possible that the secreted
ECM components contribute to the establishment of sufficient
cell-matrix interactions to maintain cell survival and
differentiation. They are also involved in the process of cell
aggregation and compaction.
[0025] All the above and other characteristics and advantages of
the invention will be further understood from the following
illustrative and non-limitative examples of preferred embodiments
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The above and other characteristics and advantages of the
invention will be more clearly understood from the detailed
description of the preferred embodiments, and from the attached
drawings in which:
[0027] FIG. 1 is a photograph of the alginate scaffold used in the
study (A) and a scanning electron micrograph of a cross-section of
the alginate scaffold (B);
[0028] FIG. 2 shows a photograph of the cell-seeded alginate
scaffolds as a function of cell seeding concentration;
[0029] FIG. 3 shows the viability of the seeded cells within the
alginate scaffolds as a function of cell seeding concentration;
.diamond-solid.10.sup.5
.tangle-solidup.2.5.times.10.sup.5.cndot.5.times.10.sup.5
.box-solid.10.sup.6 cells/scaffold;
[0030] FIG. 4 is a SEM picture of a scaffold cross-section, 4 days
after cell seeding within the scaffold;
[0031] FIG. 5 shows the immunohistochemical analysis for ECM
components of the multicellular aggregates within the
scaffolds;
[0032] FIG. 6 is a fluorescent micrograph of the seeded endothelial
cells organized into a capillary-like structure within the scaffold
in-vitro;
[0033] FIG. 7 is an histological micrograph showing the process of
capillary-like tube formation within the alginate scaffolds.
Micrographs taken after 2d (A), 7d (B) and 1-1d (C);
[0034] FIG. 8 depicts the SEM morphology of alginate scaffold
composites at .times.100 (A) and .times.400 (B) magnifications;
[0035] FIG. 9 depicts the release kinetics of VEGF from the
composite scaffolds;
[0036] FIG. 10 is a photograph of a rat heart at week 9 after
implantation of the biograft. Original magnification .times.5;
[0037] FIG. 11 is high power micrograph of hematoxylin-eosin
stained section, of the biograft 9 weeks after implantation.
Neovascularization within the biograft is indicated (L). Original
magnification .times.200;
[0038] FIG. 12 is a microscopic image of a biograft section labeled
for connexin 43, 9 weeks after implantation. Original magnification
.times.200;
[0039] FIG. 13 is a high power micrograph of hematoxylin-eosin
stained section, of the biograft 9 weeks after implantation.
Original magnification .times.100; and
[0040] FIG. 14 is a high power micrograph of hematoxylin-eosin
stained section of the biograft based on scaffold composite
containing controlled-released VEGF microspheres, 9 weeks after
implantation. Original magnification .times.100 (A) and .times.200
(B).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0041] For the purpose of clarity and as an aid in the
understanding of the invention, as disclosed and claimed herein,
the following terms and abbreviations are defined below: [0042]
MI--myocardial infarction [0043] LV--left ventricular [0044]
ECM--extracellular matrix [0045] SEM--scanning electron microscopy
[0046] VEGF--vascular endothelial growth factor [0047]
FACS--fluorescent-activated cell sorter [0048] 3-D--3-dimensional
[0049] b-FGF--basic fibroblast growth factor [0050] G--guluronic
acid
[0051] In a preferred embodiment of the invention, the fetal
cardiomyocytes or neonatal cardiomyocytes are co-cultured with
endothelial cells, cardiofibroblasts or smooth muscle cells. In yet
another preferred embodiment, said endothelial cells form
capillary-like tubes. The advantage of using endothelial cells for
capillary formation is that the capillaries formed within the 3-D
scaffold form the foundation for its vascularization after
implantation. The capillaries provide also signals such as growth
factors and extracellular matrix components to attract and enhance
blood vessel ingrowth from the host, and to improve the integration
of the functional cells. Another advantage is that in vitro
capillary formation allows a better control of the capillary
distribution inside the scaffold, as opposed to the presently
available biografts in which the implantation of scaffolds results
in an uncontrollable tissue growth into the scaffolds, which does
not leave sufficient space for the functional cells.
[0052] According to another preferred embodiment of the invention,
the polysaccharide scaffolds further comprise controlled-release
polymeric microspheres, said microspheres being able of secreting
soluble factors in a controlled manner. In another preferred
embodiment, said soluble factors comprise growth factors, genes or
DNA. Said microspheres provide a depot for soluble recombinant
factors and genes, while controlling their presentation in the
diseased tissue or graft. To maximize the effect of growth factors
on the seeded cells, the microspheres are incorporated within the
alginate scaffolds. The added advantage of using controlled-release
microspheres is that, if required, the release pattern of the
growth factor from the polymeric microspheres can be adjusted
according to specific needs. The microspheres are incorporated into
the scaffold during preparation.
[0053] Many different mammalian cell types are suitable for use in
the method of the present invention. Illustrative and
non-limitative examples of suitable cell include fetal
cardiomyocytes, neonatal cardiomyocytes, fibroblasts, smooth muscle
cells, endothelial cells, skeletal myoblasts, mesenchymal stem
cells and embryonic stem cells.
[0054] The method of the invention may be applied to the treatment
of the myocardial damage that occurs as part of many different
diseases and functional disorders. The method, however, is
particularly useful when the myocardial damage is due to myocardial
infarction. Another example is the treatment of myocardial damage
due to a congenital heart defect.
[0055] The present invention is further illustrated, but not
limited, by the following examples:
EXAMPLE 1
Tissue Engineering of a Cardiac Tissue from Fetal or Neonatal
Cardiac Cells within 3-D Alginate Scaffolds
Preparation of 3-D Alginate Scaffolds:
[0056] The 3-D scaffolds were prepared as previously described
(Shapiro and Cohen., Biomaterials, 18 583-90, 1997) from a
pharmaceutical-grade alginate, Protanal LF 5/60 (Pronova
Biopolymers, Drammen, Norway), which has a G contents (65-75%) and
solution viscosity (1% w/v, 25.degree. C.) of 50 cP. Scaffold
preparation consists of (i) Preparation of sodium alginate stock
solutions, at concentrations of 1-3% (w/v). (ii) Cross-linking of
the alginate by adding, dropwise, the bivalent cross-linker, e.g.,
calcium gluconate. (iii) Freezing the cross-linked alginate and
(iv) lyophilization to produce a sponge-like scaffold. The sponges
were sterilized using ethylene oxide gas apparatus. The residual
ethylene oxide was removed by aeration of the samples with warm air
flow. The sponges were stored in laminated bags, at room
temperature, until use. A photograph of a typical scaffold is shown
in FIG. 1A, and a scanning electon micrograph of the same scaffold
is given in FIG. 1B.
Isolation of Fetal and Neonatal Cardiomyocytes:
[0057] Fetal cardiomyocytes were isolated as described by Leor et
al., Circulation 94, II332-II336, 1996). Briefly, 15-day-old
embryos were removed from female rats and their hearts were
dissected and placed in a cold dissociation buffer (137 mM NaCl,
5.4 mM KCl, 0.8 mM MgSO.sub.4, 5.6 mM Dextrose, 0.4 mM
KH.sub.2PO.sub.4, 0.3 mM Na.sub.2HPO.sub.4, 20 mM HEPES, 500 u/ml
penicillin, 100 mg/ml streptomycin, pH 7.5). The ventricles were
cut into 1-2 mm cubes, and enzymatically-digested by Trypsin-DNAse.
Freed cells were collected in a cold M-199 culture medium,
containing 0.5% (v/v) fetal calf serum and 0.002% (w/v) DNAse,
centrifuged (0.degree. C., 10 min, 2500 rpm), washed in culture
medium and then pre-plated in a 60 mm-dish, for 15-60 min, at
37.degree. C. The nonattached cardiac cells (supernatants) were
collected and counted using a Coulter counter. Neonatal ventricular
cardiomyocytes were isolated from 2-day-old rats, as described
above. The purity of the cells (i.e, percentage cardiomyocytes) was
analyzed by Fluorescent-Activated Cell Sorter (FACS) flow
cytometry. The cells were permeabilized (15 minutes, 0.5% Triton
X-100 in PBS), incubated for 30 minutes in 1% BSA/PBS, and
subsequently incubated for 45 minutes with an antibody to
sarcomeric tropomyosin (Sigma). After washing with 0.1% Nonidet P40
and 1% BSA/PBS, the cells are incubated with FITC-conjugated goat
anti-rat IgG antibody, washed again, and were stained with
propidium iodide for DNA contents. The purity of cells was
determined Using Epics software.
Culturing of Cardiomyocytes within the 3-D Alginate Scaffolds:
[0058] The cardiomyocytes were seeded individually or cocultured
with cardiofibroblasts and/or endothelial cells, within the 3-D
alginate scaffolds. The cell ratio in the co-cultures varied as
desired. The isolated cardiac cells were seeded at a concentration,
ranging between 1.times.10.sup.5-2.times.10.sup.6 cells/scaffold,
within cylindrical alginate scaffolds (5-mm diameter.times.1.0
mm-height), placed in a 96-well plate. The cells were seeded onto
the 3-D alginate scaffolds, by a dynamic method--the centrifugal
packing method. A small volume (50-100 .mu.l) of cell suspension is
dropped on top of the scaffold or injected into the center of the
device, via a 25G needle. Immediately after overlayering the cells,
the plate containing the scaffolds is centrifuged using a
bench-type centrifuge, at 3000 rpm for 5 min. Due to their
hydrophilic nature, the alginate scaffolds were easily wetted by
the medium, and an efficient cell seeding was achieved. The seeded
constructs, supplied with additional 200 .mu.l media, were
incubated in a humidified atmosphere of 5% CO.sub.2 and 95% air, at
37.degree. C., until characterized (within 24 hours), after which
they were transferred to the cultivation bioreactor.
[0059] The efficiency of cell loading within the scaffold was
characterized within 24 hrs after cell seeding, by determining the
total cell number by quantifying the DNA content of a crude
cellular homogenate of the cardiomyocytes using the fluorescence
enhancement of 4',6-diamidino-2-phenylindole (DAPI) complexed with
DNA, as presently known in the art. The number of viable cells in
the scaffolds was evaluated using the 3-{4
3-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide (MTT)
assay, which measures the ability of mitochondrial dehydrogenase
enzymes to convert the soluble yellow MTT salt into insoluble
purple formazan salt, as presently known in the art. According to
these methods, between 85-90% of the seeded cells were efficiently
entrapped within the scaffolds. FIG. 2 shows a photograph of the
cell-seeded alginate scaffolds as a function of cell seeding
concentration, in the range of 1.times.10.sup.5 cells/scaffold to
2.times.10.sup.6 cells/scaffold. MTT assay was performed on the
different seeded scaffolds to verify cell viability and
distribution. As seen, the amount of formed purple formazan
increased with the increase in cell seeding density. The formazan
was distributed in the entire scaffolds. The viability of the
seeded cells within alginate scaffolds was maintained for almost a
month in culture, according to the MTT viability assay (FIG. 3). It
appears that the alginate scaffolds were capable of retaining the
cells and no significant cell leakage from the scaffolds was
observed.
[0060] The morphology of the cells seeded within the 3-D alginate
scaffolds was followed by scanning electron microscope (SEM). The
cell-seeded scaffolds were washed extensively with PBS, and then
fixed in 2.5% (w/v) glutaraldehyde in PBS. After washing with PBS
buffer three times, the scaffolds were dehydrated in a graded
series of water-ethanol solutions and critical-point dried. Thin
sections of the cell-seeded sponges were gold-sputtered (100
A.degree.-thickness) and examined by SEM (model JSM 35 CF, Jeol,
Japan) at 25 kV electron beam radiation. FIG. 4 is a SEM picture of
a scaffold cross-section, 4 days after cell seeding within the
scaffold. As seen, the cells were arranged as multicellular
aggregates, which were located within the scaffold pores. By a
phase contrast microscope, the cell aggregates were viable and were
contracting spontaneously and rhythmically. Thus, it appears that
the cells are forming a caridac-like tissue within the alginate
scaffolds with time.
[0061] In order to further characterize the cardiac biograft,
immunohistochemistry was used using antibodies for various
components of the extracellular matrix (ECM). Thin-sections of
cell-seeded alginate scaffolds were analyzed by an indirect
immunofluorescent assay using sheep anti-human fibronectin
(Serotec), rabbit anti-mouse laminin (ICN) and rabbit anti-human
collagen type I (ICN). The samples were frozen in liquid nitrogen
and then processed for cryostat sectioning. Frozen sections
(12.mu.-thick) were placed on microscope slides and air-dried. They
were overlaid with 0.3 ml of the specific antibody diluted at an
appropriate concentration and kept for 1 h at 37.degree. C. After
washing, the slides were incubated for 1 h in the present of the
appropriate FITC-conjugated anti-immunoglobulin antibody diluted
1:200, rinsed, and viewed under an inverted fluorescence microscope
(Olympus, Germany) equipped with a 490 nm band-pass filter with a
510-nm cutoff filter for fluorescence emission. FIG. 5 shows the
results of the immunohistochemical analysis for ECM components of
the multicellular aggregates within the scaffolds, at day 7
postseeding. They were positively stained for fibronectin, laminin
and collagen type I. It appears that the ECM was deposited on the
surface and between the individual cells that constitute the
multicellular aggregates. Control empty scaffolds, or cell-seeded
scaffolds reacted only with the second FITC-conjugated antibody,
were not stained.
EXAMPLE 2
[0062] Tissue Engineering of Capillary-Like Tubes from Endothelial
Cells Seeded within 3-D Alginate Scaffolds, In Vitro
Culturing of Endothelial Cells within the 3-D Alginate
Scaffolds:
[0063] For the isolation of Aortic Endothelial Cells, the aorta was
aseptically collected, stripped of adventitia and sliced into
rings. The rings were cultured on a tissue flask in an incubator,
at 37.degree. C., without medium. After an hour incubation, DMEM
supplemented with 10% FBS, 0.02 .mu.g/ml bFGF, 100 U/ml nystatin, 5
.mu.g/ml insulin, 5 .mu.g/ml transferrin, 5 ng/ml sodium selenite
and penicillin-streptomycin, was added. The endothelial cells grew
as colonies from the aortic rings, and then were expanded as pure
population of ondothelial cells. Purity of the cells was analyzed
by FACS using anti-von Willebrant Factor antibodies. The
endothelial cells were seeded onto the alginate scaffolds at an
initial cell density of 1 million per scaffold. The growth medium
was supplemented with rHuVEGF165 VEGF (50 ng/ml) (produced using
baculovirus recombinant system and affinity-purified to yield the
native disulfide linked dimer (45 kDa)). The organization and
viability of the cells was followed by a fluorescent double
staining technique. This technique uses DiOC18, which stains
membranes of viable cells in green and Propidium iodide, which
stains in red the nuclei of dead cells. Within 2 weeks in culture
the seeded endothelial cells were organized into a cord-like
structure within the scaffold in vitro (FIG. 6). Nearly all the
cells were viable as they were all stained only in green. In
addition, the two cords were not on the same plane, indicating the
presence of a three-dimensional structure.
[0064] To verify the existence of a lumen in the cords, a
gelatin-paraffin double embedding technique was used. The biografts
were fixed in 10% neutral buffered formalin for 30 min, washed
three times in DMEM solution and incubated for 1 hr in gelatin
solution. The biografts in gelatin were cooled at 40.degree. C. for
5 min, and transferred to 10% neutral buffered formalin for
overnight incubation. The biografts were horizontally sliced (3-4
mm thick slices), then placed in a processing cassette and paraffin
embedded. 6 .mu.m-thick slices were cut and air dried for 2 days in
an incubator at 37.degree. C. The slices were immunostained using
rabbit-anti human von willebrand factor (factor VIII)-IgG, which
reacts specifically with cytoplasm of endothelial cells. As a
counter staining, the cells were stained with hematoxylin. FIG. 7
demonstrates the process of cord formation within the alginate
scaffolds. At first, the cells were organized into multicellular
aggregates within two days in culture (FIG. 7A). The cell aggregate
was positively stained with Anti-factor VIII, indicating that the
endothelial nature of cells is maintained. After one week, it was
possible to identify further organization of the cells composing
the aggregate (FIG. 7B). The endothelial cells at the periphery of
the aggregate were organized in a row. Two weeks after the cell
seeding the formation of rings could be identified (FIG. 7C). Rings
composed from endothelial cells were found in successive serial
sections, indicating that the tissue-engineered tubes have a
lumen.
EXAMPLE 3
Preparation of Composite Alginate Scaffolds Containing Microspheres
with Controlled Release Growth Factors
[0065] The microspheres containing the growth factors are prepared
from poly(D,L-Lactide-(o-glycolide) (PLGA) (RG502H, Boehringer
Ingelheim, Germany) by the solvent evaporation method, based on a
double emulsion (Cohen et al., Pharmaceutical Research, 8, 713-720,
1991). The polymer was dissolved in a volatile organic solvent,
methylene chloride. An aqueous solution containing the growth
factors was added to the polymer solution, and the mixture was
homogenized to create an inner emulsion. This emulsion was further
emulsified in a second aqueous phase that contained a surface
active agent such as poly (vinyl alcohol). The resulting double
emulsion was stirred until all organic solvent was evaporated,
leaving solid microspheres. To formulate the composite scaffold,
the microspheres were suspended in the alginate solution, and
scaffold preparation proceeds as described in Example 1.
[0066] The alginate scaffold composites displayed a highly porous
structure (>90% porosity), with uniformly distributed pore size
of 100 .mu.m. The incorporation of the 5 .mu.m-diameter PLGA
microspheres during scaffold preparation did not affect its porous
morphology. FIG. 8 depicts the SEM morphology of alginate scaffold
composites at different magnifications. The microspheres appeared
to be evenly distributed throughout the construct (FIG. 8A).
Analysis at higher magnification revealed that the microspheres are
in fact an integral part of the scaffold wall (FIG. 8B), and do not
interfere with pore structure. FIG. 9 depicts the release
characteristics of VEGF from the composite scaffolds. The
concentration of VEGF in the medium was determined using an
enzyme-linked immuno assay (ELISA). As seen, the release pattern
was characterized with a continuous protein release for over a
month. When compared to the degradation kinetics of these
microspheres, it appears that the microspheres released the growth
factors in both diffusion and degradation-dependent manner.
EXAMPLE 4
Biograft Transplantation within the Infarcted Myocardium
[0067] Myocardial infarction was induced in female Sprague-Dawley
rats by permanent occlusion of the left main coronary artery by an
intramural stitch (Leor et al., Circulation, 94 (suppl II):
II332-II336, 1996). The experimental group (n=6) was treated with
biograft transplantation and the control group (n=6) was treated
with sham transplantation (insertion of one suture into the
myocardial scar). Biograft transplantation and sham transplantation
were performed 7 days after MI. Rats were anesthetized and the
chest was opened under sterile conditions. The infarcted area was
identified visually by surface scar and wall motion abnormality.
Rats were randomized to implantation of biografts or sham
transplantation into the infarcted myocardium. Two scaffolds were
attached, by one suture for each, to the scar. Air was expelled
from the chest and the surgical incision sutured closed.
EXAMPLE 5
Intense Neovascularization and and Myofiber Formation within the
Implanted Biograft, 9 Weeks Post Implantation
[0068] Visual inspection of the implanted biograft, 65.+-.5 days
post implantation (as described in example 4), revealed that the
scaffold was covered by a thin connective tissue enriched with
blood vessels (FIG. 10). The extensive neovascularization into the
biograft emerged from the neighboring coronary network (B). In
addition, a coronary branch (C) that supplies the biograft covered
it with an extensive network of vessels.
[0069] Adjacent blocks of the harvested heart were embedded in
paraffin, sectioned into 5-.mu.m slices and stained for
hematoxylin/eosin. Histologic examination of thin sections of the
biograft identified differentiated forms of myocardial tissue (FIG.
11). Well-formed myofibers with typical striation were found to
grow in between collagen bundles. Some myofibers displayed the
normal parallel arrangements of cardiomyocytes, while others were
randomly oriented. Significantly, the biografts were populated with
newly-formed capillaries and arterioles (L), embedded within the
collagen bundle matrix.
EXAMPLE 6
Microscopic Analysis of Biograft Labeled for Connexin 43
[0070] Serial sections of the paraffin embedded tissue blocks
(prepared as described in example 5), were immunolabeled with
antibodies against the gap junctional protein, connexin 43 (Kanter
et al., Circ Res, 72:1124-1131, 1993). Connexin 43 was localized in
the normal parallel arrangements in the host myocardium (H in FIG.
12) and randomly oriented in the biograft (B). The presence of
cellular gap junctions in these preparations (FIG. 12), indicates
the presence of mechanical and electrical connections among the
cardiomyocytes in the graft.
EXAMPLE 7
Tissue Ingrowth and Graft Integration with the Host Myocardium
[0071] Histological staining of thin sections of the biograft, 9
weeks post implantation (prepared from the tissue blocks described
in example 5), revealed tissue ingrowth characterized by the
presence of fibrous strands of collagen (FIG. 13). At many
anchorage sites, the biograft showed integration with the host
myocardium (H) and the specimens showed almost complete
disappearance of the scaffold.
[0072] When tissue-engineered biografts based on the alginate
scaffold composites containing VEGF and/or bFGF (see Example 3),
were implanted in the infarct scar, the localized release of the
growth factors within the scaffold enhanced the vascularization of
the scaffold and integration of the biograft into the infarcted
myocardium (FIG. 14).
EXAMPLE 8
Echocardiography to Evaluate Remodeling and Contractility
[0073] Echocardiography was performed in order to evaluate the
influence of the biografts on left ventricular remodeling and
function. Transthoracic echocardiography was performed on both
experimental and sham animals (treated as described in example 4),
5-7 days after MI, prior to transplantation (baseline
echocardiogram), and 65.+-.5 days after transplantation of the
biograft (in the experimental group), and after the insertion of
the suture (in the sham group). Briefly, rats were anesthetized
with ketamine 50 mg/kg and xylasine 10 mg/kg. The chest was shaved,
and the rats were placed supine. Echocardiograms were performed
with a commercially available echocardiography system equipped with
7.5-MHz phased-array transducer (Hewlett Packered, Andover, Mass).
The transducer was positioned on the left anterior side of the
chest after the precordium was shaved. The heart was first imaged
in 2-D mode in the parasternal long axis and short axis views of
the left ventricle. By the use of these views, the M-mode cursor
was positioned perpendicular to the ventricular septum and
posterior wall; M-mode images were then obtained at the level below
the tip of the mitral valve leaflets at the level of the papillary
muscles. Care was taken to avoid excessive pressure. Posterior wall
thickness and LV internal dimensions were measured according to the
leading edge method of the American Society of Echocardiography.
Maximal LV end-diastolic dimension (at the time of maximal cavity
dimension); minima left ventricular end-systolic dimension (at the
time of maximum anterior motion of the posterior wall); and
fractional shortening as a measure of systolic function calculated
as % FS=[(LVIDd-LVIDs)/LVIDs]/.times.100, where LVID indicates left
ventricular internal dimension, s indicates systole, and d
indicates diastole. To further validate these measurements and to
ascertain the accuracy and reproducibility of the technique, we
carried out a reproducibility study in normal rats. All
measurements were averaged on three consecutive cardiac cycles and
performed by an experienced technician blinded to the treatment
group. The statistical significance of differences between
measurements before and after transplantation was assessed by use
of the paired t test.
[0074] The sham group developed a typical course of LV remodeling
and heart failure complicating anterior MI. After 3 months, LV
end-diastolic and systolic internal diameters increased
progressively, by 31% and 65% respectively (Table IA). Progressive
LV dilatation was also accompanied by significant deterioration in
LV performance, shown by the deterioration of fractional shortening
(from 47.+-.2% at baseline to 33.+-.4%; p=0.005) at the end of the
study.
[0075] Conversely, in the biograft-treated rats, attenuation of all
LV remodeling indices was observed (Table IB). During the follow-up
period, there was no significant change in the LV internal
diastolic and systolic diameters (0.64.+-.0.04 vs. 0.69.+-.0.02 cm;
p=0.31, and 0.32.+-.0.04 vs. 0.37=0.04 cm; p=0.52, respectively).
The beneficial effect of the biografts on LV remodeling is seen in
the prevention of LV function deterioration, as reflected by
preservation of fractional shortening after implantation (53.+-.4
vs. 47.+-.5%, p=0.52). TABLE-US-00001 TABLE I A. Sham group
Baseline 9 wks after echocardiogram suture insertion P LV internal
diameter (cm): End diastole 0.64 .+-. 0.03 0.84 .+-. 0.05 0.03 End
systole 0.33 .+-. 0.02 0.55 .+-. 0.06 0.02 Fractional 47 .+-. 2 33
.+-. 4 0.005 shortening (%) LV wall thickness (cm): Anterior 0.10
.+-. 0.004 0.09 .+-. 0.01 0.34 Posterior 0.12 .+-. 0.006 0.13 .+-.
0.002 0.07 B. Biograft-treated group 9 wks after Baseline biograft
echocardiogram transplantation P LV internal diameter (cm): End
diastole 0.64 .+-. 0.04 0.69 .+-. 0.02 0.32 End systole 0.32 .+-.
0.04 0.37 .+-. 0.04 0.52 Fractional 53 .+-. 4 47 .+-. 5 0.52
shortening (%) LV wall thickness (cm): Anterior 0.09 .+-. 0.004
0.10 .+-. 0.007 0.21 Posterior 0.11 .+-. 0.006 0.12 .+-. 0.007
0.44
[0076] While specific embodiments of the invention have been
described for the purpose of illustration, it will be understood
that the invention may be carried out in practice by skilled
persons with many modifications, variations and adaptations,
without departing from its spirit or exceeding the scope of the
claims.
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