U.S. patent application number 12/981260 was filed with the patent office on 2011-09-01 for methods for using a three-dimensional stromal tissue to promote angiogenesis.
This patent application is currently assigned to Theregen, Inc.. Invention is credited to Jonathan Noel Mansbridge, Gail K. Naughton, Robert Emmett Pinney, Joan Zeltinger.
Application Number | 20110213470 12/981260 |
Document ID | / |
Family ID | 26826992 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110213470 |
Kind Code |
A1 |
Naughton; Gail K. ; et
al. |
September 1, 2011 |
METHODS FOR USING A THREE-DIMENSIONAL STROMAL TISSUE TO PROMOTE
ANGIOGENESIS
Abstract
The present invention relates to a method for promoting blood
vessel formation in tissues and organs. In particular, the method
relates to implantation or attachment of an engineered
three-dimensional stromal tissue to promote endothelialization and
angiogenesis in the heart and related tissues. The
three-dimensional stromal tissue of the present invention may be
used in a variety of applications including, but not limited to,
promoting repair of and regeneration of damaged cardiac muscle,
promoting vascularization and healing during cardiac surgery,
promoting blood vessel formation at anastomosis sites, and
promoting vascularization and repair of damaged skeletal muscle,
smooth muscle or connective tissue.
Inventors: |
Naughton; Gail K.; (San
Diego, CA) ; Mansbridge; Jonathan Noel; (La Jolla,
CA) ; Pinney; Robert Emmett; (Poway, CA) ;
Zeltinger; Joan; (San Diego, CA) |
Assignee: |
Theregen, Inc.
San Francisco
CA
|
Family ID: |
26826992 |
Appl. No.: |
12/981260 |
Filed: |
December 29, 2010 |
Related U.S. Patent Documents
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12420716 |
Apr 8, 2009 |
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12981260 |
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10851938 |
May 21, 2004 |
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12420716 |
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09411585 |
Oct 1, 1999 |
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10851938 |
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60128838 |
Apr 12, 1999 |
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Current U.S.
Class: |
623/23.72 |
Current CPC
Class: |
A61P 9/10 20180101; A61L
27/507 20130101; A61L 27/3839 20130101; A61P 43/00 20180101; A61L
27/3804 20130101; A61L 27/3886 20130101; A61P 41/00 20180101 |
Class at
Publication: |
623/23.72 |
International
Class: |
A61F 2/02 20060101
A61F002/02 |
Claims
1-41. (canceled)
42. A method for effecting angiogenesis in ischemic heart tissue of
a subject comprising contacting ischemic heart tissue of said
subject with a three-dimensional stromal tissue comprising
fibroblasts and connective tissue proteins naturally secreted by
the fibroblasts, said cells being attached to and substantially
enveloping a framework composed of a biocompatible, non-living
material formed into a three-dimensional structure having
interstitial spaces bridged by the fibroblasts; wherein said
stromal tissue is capable of effecting angiogenesis in ischemic
heart tissue.
43. The method of claim 42 wherein the framework is composed of a
biodegradable material.
44. The method of claim 43 wherein the biodegradable material is
polyglycolic acid, catgut sutures, cellulose, gelatin, collagen, or
dextran.
45. The method of claim 42 wherein the framework is a mesh.
46. The method of claim 42 wherein the stromal tissue is obtained
directly from a fresh culture.
47. The method of claim 42 wherein the stromal tissue has been
cryopreserved.
48. The method of claim 42 further comprising adhering the stromal
tissue to the ischemic heart tissue by natural cellular
attachment.
49. The method of claim 42 further comprising attaching the stromal
tissue to the ischemic heart tissue by means of a biodegradable or
non-biodegradable suture, a biologic glue, a synthetic glue, a
laser dye, or a hydrogel.
50. The method of claim 42 wherein the ischemic tissue is of the
heart epicardium.
51. A method for effecting vascularization in damaged cardiac
tissue of a subject, comprising attaching a three-dimensional
stromal tissue to an epicardial or myocardial surface near a
damaged region of cardiac tissue of a subject, wherein the
three-dimensional stromal tissue comprises fibroblasts and
connective tissue proteins naturally secreted by the fibroblasts,
said cells being attached to and substantially enveloping a
framework composed of a biocompatible, non-living material formed
into a three-dimensional structure having interstitial spaces
bridged by the fibroblasts; and wherein said stromal tissue is
capable of effecting vascularization in damaged cardiac tissue.
52. The method of claim 51, wherein the damaged region is
ischemic.
53. The method of claim 51, wherein said attaching comprises
attaching the stromal tissue to the damaged region by means of a
biodegradable or non-biodegradable suture, a biologic glue, a
synthetic glue, a laser dye, or a hydrogel.
Description
1. INTRODUCTION
[0001] The present invention relates to a method for promoting,
angiogenesis in organs and tissues. In particular, the method
relates to implantation or attachment of a three-dimensional
stromal tissue to promote endothelialization and vascularization in
the heart and related tissues.
2. BACKGROUND OF THE INVENTION
[0002] Coronary heart disease is the single leading cause of death
in America today (American Heart Association's "1999 Heart and
Stroke Statistical Update"). This disease, as with various other
cardiovascular disorders, is characterized by the narrowing of
arteries and inadequate blood flow to critical tissues.
[0003] Currently used clinical methods for improving blood flow in
a diseased or otherwise damaged heart involve invasive surgical
techniques such as coronary by-pass surgery, angioplasty, and
endarterectomy. Such procedures naturally involve high-degrees of
inherent risk during and after surgery, and often only provide a
temporary remedy to cardiac ischemia.
[0004] In an effort to improve the prognosis of surgical procedures
on the heart, physicians and researchers have attempted to use
pumps to assist blood flow during surgery. However, such pumps only
act as temporary assist devices during surgery, they cannot be used
as a form of treatment for the cardiac condition.
[0005] An alternative, or at least a compliment, to coronary
by-pass and other surgical procedures to improve blood flow in the
heart is to induce tissues in the heart to form new blood vessels.
In that regard, angiogenic compounds such as vascular endothelial
growth factor (VEGF), have been used in an effort to facilitate the
formation of new blood vessels. One approach to using VEGF to
promote blood vessel formation in heart tissue has been to inject
the protein directly into a patient's body. However, such attempts
have been largely unsuccessful.
[0006] Recently, a gene-therapy approach was used to deliver VEGF
by injection of retroviral vectors that targeted heart tissue and
resulted in VEGF production (Losordo et al., 1998, Circulation
98:2800-2804). This in situ method improved blood flow and
subjective symptoms in patients, suggesting that local delivery of
a growth factor such as VEGF to promote angiogenesis in heart
tissues may be of therapeutic value in the treatment of certain
heart conditions. However, such gene therapy techniques utilizing
retroviral vectors present certain inherent risks and safety
concerns. In addition, gene therapy-type approaches present a
number of unresolved, problematic technical hurdles such as low
transfection levels for recipient cells, construct instability and
long-term expression of the desired gene product from the
transfected cells.
3. SUMMARY OF TEE INVENTION
[0007] The present invention relates to a method for promoting
blood vessel formation in tissues and organs. In particular, the
method relates to implantation or attachment of a three-dimensional
stromal tissue to promote endothelialization and angiogenesis in
the heart and related tissues.
[0008] The invention has a variety of applications including, but
not limited to, promoting repair of and regeneration of damaged
cardiac muscle, promoting vascularization and healing during
cardiac surgery (e.g. by-pass surgery or heart valve replacement),
promoting blood vessel formation at anastomosis sites, and
promoting vascularization and healing of ischemic or otherwise
damaged tissues such as skeletal muscle, smooth muscle, brain
tissue or connective tissue.
[0009] The invention is based in part on the discovery that
three-dimensional stromal tissue constructs, when implanted in the
wound bed of patients with diabetic foot ulcers, are capable of
inducing rapid endothelialization and vascularization, resulting in
new capillary formation and reduced inflammation in the wounded
tissue.
[0010] The three-dimensional stromal tissue implants secrete a
variety of growth factors critical to tissue regeneration and
angiogenesis, most notably vascular endothelial growth factor, or
VEGF (Table II). The invention encompasses the application of the
three-dimensional stromal tissue to damaged tissues, such as
damaged cardiac muscle, to induce a new local blood supply to the
area and support rapid tissue remodeling.
[0011] A three-dimensional stromal tissue implant may also be used
to promote formation of a "natural" carotid by-pass to assist in,
or obviate the need for, carotid endarterectomy surgery (which can
often result in stroke due to downstream flow of particles
dislodged during the procedure).
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A-1D Photomicrographs showing engineered stromal
tissue-stimulated angiogenesis in a chick chorioallantoic
membrane(CAM). FIGS. 1A and 1B show macroscopic view, while FIGS.
1C and 1D show histology. FIG. 1A shows scaffold alone, FIG. 1C
shows non-viable, and FIGS. 1B and 1D show Three-dimensional
stromal tissue treated membrane.
[0013] FIG. 2 Bar graph depicting the effect of engineered stromal
tissue on capillary blood vessel formation in a chick
chorioallantoic membrane. Bars represent 95% confidence
intervals.
[0014] FIG. 3 Bar graph depicting blood vessel formation stimulated
by engineered stromal tissue in a rat aortic ring assay.
[0015] FIG. 4 Bar graph depicting proliferation of human umbilical
vein endothelial cell (HUVEC) in vitro following stimulation by
engineered stromal tissue conditioned medium.
[0016] FIG. 5 Bar graph depicting stimulation of endothelial cell
motility by engineered stromal tissue.
[0017] FIG. 6 Graphs depicting stimulation of endothelial cell
chemotaxis by engineered stromal tissue (left side shows standard
curve using purified VEGF at indicated concentrations).
[0018] FIG. 7 Flow cytometry result depicting induction of integrin
.alpha.,.beta., expression on endothelial cells by engineered
stromal tissue.
[0019] FIGS. 8A and 8B Photomicrographs depicting inhibition of
endothelial cell apoptosis cultured on "MATRIGEL" in the presence
of engineered stromal tissue. The cells were stained with low
density lipoprotein and sytox, which stained nuclei of apoptotic
cells.
[0020] FIG. 9A-9D Photomicrographs of a human diabetic ulcer
showing engineered stromal tissue-stimulated vascularization of the
wound bed, remodeling of the tissue, and reduction in inflammation.
FIGS. 9A and 9C show the wound bed before treatment, while FIGS. 9B
and 9D show the wound bed after treatment.
5. DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a method for promoting
blood vessel formation in tissues and organs of a subject,
particularly a human subject. In particular, the method relates to
implantation or attachment of an engineered three-dimensional
stromal tissue to promote endothelialization and angiogenesis in
the heart and related tissues.
[0022] The invention has a variety of applications including, but
not limited to, promoting repair of and regeneration of damaged
cardiac muscle, promoting vascularization and healing during
cardiac surgery (e.g. by-pass surgery or heart valve replacement),
promoting blood vessel formation at anastomosis sites, and
promoting vascularization and repair of damaged skeletal muscle,
connective tissue, or other tissues.
[0023] The invention is based, in part, on the discovery that
three-dimensional stromal tissues, when implanted in the wound bed
of patients with diabetic foot ulcers, are capable of inducing
endothelialization and vascularization, resulting in new capillary
formation and reduced inflammation in the wounded tissue.
[0024] The three-dimensional stromal tissue comprises stromal cells
grown on a three-dimensional substrate or framework composed of a
biocompatible, non-living material formed into a three-dimensional
structure having interstitial spaces bridged by the stromal cells.
The stromal cells preferably comprise fibroblasts with or without
additional cells and/or elements described more fully herein below.
In particular, the additional cells may comprise smooth muscle
cells, cardiac muscle cells, endothelial cells or skeletal muscle
cells. The fibroblasts and/or other cells may be fetal or adult in
origin, and may be derived from convenient sources such as skin,
cardiac muscle, smooth muscle, skeletal muscle, liver, pancreas,
brain etc. Such tissues and or organs can be obtained by
appropriate biopsy or upon autopsy. In fact, cadaver organs may be
used to provide a generous supply of stromal cells and
elements.
[0025] It is to be understood that one skilled in the art can
control the angiogenic activity of a stromal tissue culture by
incorporating cells that release different levels of angiogenic
factors. For example, vascular smooth muscle cells, preferably
aortic smooth muscle cells, are known to produce substantially more
VEGF than human dermal fibroblasts. Therefore, by utilizing aortic
smooth muscle cells instead of or in addition to fibroblasts, one
can culture three-dimensional stromal tissues with enhanced
angiogenic activity.
[0026] In an alternative embodiment of the invention, a
three-dimensional stromal tissue implant that is genetically
engineered to have improved properties for inducing angiogenesis
may be used to promote formation of new blood vessels in the heart
or other tissues.
[0027] In another embodiment, the invention encompasses a method of
treatment of ischemic damage to heart, brain, visceral organs or
peripheral tissues. For example, and not by way of limitation, one
embodiment of the invention entails attaching a three-dimensional
stromal tissue to an ischemic region of a heart following
myocardial infarction to promote vascularization of the heart and
regeneration of damaged cardiac muscle cells. In the case of
cerebral ischemia (e.g. resulting from a stroke and/or elevated
intracranial pressure) the three dimensional stromal tissue implant
may include fibroblasts, neural glial cells, neural stem cells,
astrocytes, fibroblasts transfected with nerve growth factor, or a
combination thereof. Such a stromal tissue implant is placed
directly on the cerebral cortex or surgically implanted in the
region of ischemia.
[0028] In yet another embodiment, the invention encompasses
application of the three-dimensional stromal tissue to any tissue
or organ to promote angiogenesis with the proviso that the organ or
tissue is not a diabetic foot ulcer or a veinous ulcer.
5.1 Preparation of a Three-Dimensional Stromal Tissue
[0029] For the practice of the present invention, stromal cells are
inoculated upon a three-dimensional framework, and grown to develop
a stromal tissue. The three-dimensional support framework may be of
any material and/or shape that: (a) allows cells to attach to it
(or can be modified to allow cells to attach to it); and (b) allows
cells to grow in more than one layer. Alternatively, a
substantially two-dimensional sheet or membrane may be used to
culture monolayers of cells.
[0030] A number of different materials may be used to form the
framework, including but not limited to: nylon (polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl
compounds (e.g., polyvinylchloride; PVC), polycarbonate,
polytetrafluorethylene (PTFE; TEFLON), thermanox (TPX),
nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures,
cellulose, gelatin, dextran, etc. Any of these materials may be
woven into a mesh to form the three-dimensional framework. Certain
materials, such as nylon, polystyrene, etc., are poor substrates
for cellular attachment. When these materials are used as the
three-dimensional support framework, it is advisable to pre-treat
the framework prior to inoculation of stromal cells in order to
enhance the attachment of stromal cells to the framework. For
example, prior to inoculation with stromal cells, nylon screens
could be treated with 0.1 M acetic acid, and incubated in
polylysine, fetal bovine serum, and/or collagen to coat the nylon.
Polystyrene could be similarly treated using sulfuric acid.
[0031] When the three-dimensional stromal tissue is to be implanted
directly in vivo, it may be preferable to use biodegradable
materials such as PGA, catgut suture material, collagen, polylactic
acid, or hyaluronic acid. For example, these materials may be woven
into a three-dimensional framework such as a collagen sponge or
collagen gel. Where the cultures are to be maintained for long
periods of time or cryopreserved, non-degradable materials such as
nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons,
cotton, etc. may be preferred. A convenient nylon mesh which could
be used in accordance with the invention is Nitex, a nylon
filtration mesh having an average pore size of 140 .mu.m and an
average nylon fiber diameter of 90 .mu.m (#3-210/36, Tetko, Inc.,
N.Y.).
[0032] Stromal cells comprising fibroblasts, with or without other
cells and elements described below, are inoculated onto the
framework. These stromal cells may be derived from tissues or
organs, such as skin, heart, blood vessels, skeletal muscle, liver,
pancreas, brain etc., which can be obtained by biopsy (where
appropriate) or upon autopsy. In fact, fibroblasts and other
stromal cells can be obtained in quantity rather conveniently from
any appropriate cadaver organ. As previously explained, fetal
fibroblasts can be used to form a "generic" three-dimensional
stromal tissue that will support the growth of a variety of
different cells and/or tissues that come in contact with it.
However, a "specific" stromal tissue may be prepared by inoculating
the three-dimensional framework with stromal cells derived from the
heart and/or from a particular individual who is later to receive
the cells and/or tissues grown in culture in accordance with the
three-dimensional culture of the invention.
[0033] Stromal cells may be readily isolated by disaggregating an
appropriate organ or tissue. This may be readily accomplished using
techniques known to those skilled in the art. For example, the
tissue or organ can be disaggregated mechanically and/or treated
with digestive enzymes and/or chelating agents that weaken the
connections between neighboring cells making it possible to
disperse the tissue into a suspension of individual cells without
appreciable cell breakage. Enzymatic dissociation can be
accomplished by mincing the tissue and treating the minced tissue
with any of a number of digestive enzymes either alone or in
combination. These include, but are not limited to, trypsin,
chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase,
pronase, dispase etc. Mechanical disruption can also be
accomplished by a number of methods including, but not limited to,
the use of grinders, blenders, sieves, homogenizers, pressure
cells, or insonators to name but a few. For a review of tissue
disaggregation techniques, see Freshney, Culture of Animal Cells. A
Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York,
1987, Ch. 9, pp. 107-126.
[0034] Once the tissue has been reduced to a suspension of
individual cells, the suspension can be fractionated into
subpopulations from which the fibroblasts and/or other stromal
cells and/or elements can be obtained. This also may be
accomplished using standard techniques for cell separation
including, but not limited to, cloning and selection of specific
cell types, selective destruction of unwanted cells (negative
selection), separation based upon differential cell agglutinability
in the mixed population, freeze-thaw procedures, differential
adherence properties of the cells in the mixed population,
filtration, conventional and zonal centrifugation, centrifugal
elutriation (counter-streaming centrifugation), unit gravity
separation, countercurrent distribution, electrophoresis and
fluorescence-activated cell sorting. For a review of clonal
selection and cell separation techniques, see Freshney, Culture of
Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss,
Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.
[0035] The isolation of stromal cells may, for example, be carried
out as follows: fresh tissue samples are thoroughly washed and
minced in Hanks balanced salt solution (HESS) in order to remove
serum. The minced tissue is incubated from 1-12 hours in a freshly
prepared solution of a dissociating enzyme such as trypsin. After
such incubation, the dissociated cells are suspended, pelleted by
centrifugation and plated onto culture dishes. All stromal cells
will attach before other cells, therefore, appropriate stromal
cells can be selectively isolated and grown. The isolated stromal
cells can then be grown to confluency, lifted from the confluent
culture and inoculated onto the three-dimensional framework (U.S.
Pat. No. 4,963,489; Naughton et al., 1987, J. Med.
18(3&4):219-250). Inoculation of the three-dimensional
framework with a high concentration of stromal cells, e.g.,
approximately 10.sup.6 to 5.times.10.sup.7 cells/ml, will result in
the establishment of the three-dimensional stromal tissue in
shorter periods of time.
[0036] In addition to fibroblasts, other cells may be added to form
the three-dimensional stromal tissue required to support long term
growth in culture. For example, other cells found in loose
connective tissue may be inoculated onto the three-dimensional
framework along with, or instead of, fibroblasts. Such cells
include but are not limited to endothelial cells, pericytes,
macrophages, monocytes, adipocytes, skeletal muscle cells, smooth
muscle cells, cardiac muscle cells, etc. Such cells may be
inoculated onto the three-dimensional framework in the absence of
fibroblasts. These stromal cells may readily be derived from
appropriate tissues or organs such as skin, heart, blood vessels,
etc., using methods known in the art such as those discussed above.
In a specific embodiment of the invention, fibroblasts are
inoculated onto the framework.
[0037] It is to be understood that one skilled in the art can
control the angiogenic activity of a stromal tissue culture by
incorporating cells that release different levels of angiogenic
factors. For example, vascular smooth muscle cells, preferably
aortic smooth muscle cells, are known to produce substantially more
VEGF than human dermal fibroblasts. Therefore, by utilizing aortic
smooth muscle cells instead of or in addition to fibroblasts, one
can culture three-dimensional stromal tissues with enhanced
angiogenic activity.
[0038] Again, where the cultured cells are to be used for
transplantation or implantation in vivo, it is preferable to obtain
the stromal cells from the patient's own tissues. The growth of
cells in the presence of the three-dimensional stromal support
framework may be further enhanced by adding to the framework, or
coating it with proteins (e.g., collagens, elastin fibers,
reticular fibers) glycoproteins, glycosaminoglycans (e.g., heparan
sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan
sulfate, keratan sulfate, etc.), a cellular matrix, and/or other
materials.
[0039] After inoculation of the stromal cells, the
three-dimensional framework should be incubated in an appropriate
nutrient medium. Many commercially available media such as RPMI
1640, Fisher's, Iscove's, McCoy's, and the like may be suitable for
use. It is important that the three-dimensional stromal tissue be
suspended in the medium during the incubation period in order to
maximize proliferative activity. In addition, the culture should be
"fed" periodically to remove the spent media, depopulate released
cells, and add fresh media. During the incubation period, the
stromal cells will grow linearly along and envelop the filaments of
the three-dimensional framework before beginning to grow into the
openings of the framework.
[0040] The openings of the framework should be of an appropriate
size to allow the stromal cells to stretch across the openings.
Maintaining actively growing stromal cells which stretch across the
framework enhances the production of growth factors which are
elaborated by the stromal cells, and hence will support long term
cultures. For example, if the openings are too small, the stromal
cells may rapidly. achieve confluence but be unable to easily exit
from the mesh; trapped cells may exhibit contact inhibition and
cease production of the appropriate factors necessary to support
proliferation and maintain long term cultures. If the openings are
too large, the stromal cells may be unable to stretch across the
opening; this will also decrease stromal cell production of the
appropriate factors necessary to support proliferation and maintain
long term cultures. When using a mesh type of framework, as
exemplified herein, it has been found that openings ranging from
about 140 .mu.m to about 220 .mu.m will work satisfactorily.
However, depending upon the three-dimensional structure and
intricacy of the framework, other sizes may work equally well. In
fact, any shape or structure that allows the stromal cells to
stretch and continue to replicate and grow for lengthy time periods
will work in accordance with the invention.
[0041] Different proportions of the various types of collagen
deposited on the framework can affect the growth of the cells that
come in contact with the three dimensional stromal tissue. The
proportions of extracellular matrix (ECM) proteins deposited can be
manipulated or enhanced by selecting fibroblasts which elaborate
the appropriate collagen type. This can be accomplished using
monoclonal antibodies of an appropriate isotype or subclass that is
capable of activating complement, and which define particular
collagen types. These antibodies and complement can be used to
negatively select the fibroblasts which express the desired
collagen type. Alternatively, the stroma used to inoculate the
framework can be a mixture of cells which synthesize the
appropriate collagen types desired. The distribution and origins of
the various types of collagen is shown in Table I.
TABLE-US-00001 TABLE I DISTRIBUTIONS AND ORIGINS OF VARIOUS TYPES
OF COLLAGEN Collagen Principal Type Tissue Distribution Cells of
Origin I Loose and dense ordinary Fibroblasts and. connective
tissue; reticular cells; collagen fibers smooth muscle cells
Fibrocartilage Bone Osteoblast Dentin Odontoblasts II Hyaline and
elastic Chondrocytes cartilage Vitreous body of eye Retinal cells
III Loose connective tissue; Fibroblasts and reticular fibers
reticular cells Papillary layer of dermis Blood vessels Smooth
muscle cells; endothelial cells IV Basement membranes Epithelial
and endothelial cells Lens capsule of eye Lens fibers V Fetal
membranes; Fibroblast placenta Basement membranes Bone Smooth
muscle Smooth muscle cells VI Connective tissue Fibroblasts VII
Epithelial basement Fibroblasts, membranes, anchoring keratinocytes
fibrils VIII Cornea Corneal fibroblasts IX Cartilage X Hypertrophic
cartilage XI Cartilage XII Papillary dermis Fibroblasts XIV,
Reticular dermis Fibroblasts undulin XVII P170 bullous pemphigoid
Keratinocytes antigen
Thus, since the three-dimensional culture system described herein
is suitable for the growth of diverse cell types and tissues, and
depending upon the tissue to be cultured and the collagen types
desired, the appropriate stromal cell(s) may be selected to
inoculate the three-dimensional framework.
[0042] During incubation of the three-dimensional stromal support,
proliferating cells may be released from the framework. These
released cells may stick to the walls of the culture vessel where
they may continue to proliferate and form a confluent monolayer.
This should be prevented or minimized, for example, by removal of
the released cells during feeding, or by transferring the
three-dimensional stromal tissue to a new culture vessel. The
presence of a confluent monolayer in the vessel may "shut down" the
growth of cells in the three-dimensional culture. Removal of the
confluent monolayer or transfer of the stromal tissue to fresh
media in a new vessel will restore proliferative activity of the
three-dimensional culture system. Such removal or transfers should
be done in any culture vessel which has a stromal monolayer
exceeding 25% confluency. Alternatively, the culture system could
be agitated to prevent the released cells from sticking, or instead
of periodically feeding the cultures, the culture system could be
set up so that fresh media continuously flows through the system.
The flow rate could be adjusted to both maximize proliferation
within the three-dimensional culture, and to wash out and remove
cells released from the culture, so that they will not stick to the
walls of the vessel and grow to confluence. In any case, the
released stromal cells can be collected and cryopreserved for
future use.
5.2 Preparation of a Genetically Engineered Three-Dimensional
Stromal Tissue
[0043] Genetically engineered three-dimensional stromal tissue may
be prepared as described in U.S. Pat. No. 5,785,964 which is
incorporated herein by reference. A genetically-engineered stromal
tissue may serve as a gene delivery vehicle for sustained release
of angiogenic factors in vivo.
[0044] Stromal cells may be engineered to express an exogenous gene
product. Stromal cells that can be genetically engineered include,
but are not limited to, fibroblasts, smooth muscle cells, cardiac
muscle cells, mesenchymal stem cells, and other cells found in
loose connective tissue such as endothelial cells, macrophages,
monocytes, adipocytes, pericytes, reticular cells found in bone
marrow, etc.
[0045] The cells and tissues may be engineered to express a target
gene product which may impart a wide variety of functions,
including, but not limited to, enhanced function of the genetically
engineered cells and tissues to promote angiogenesis when implanted
in vivo. The target gene product may be a peptide or protein, such
as an enzyme, hormone, cytokine, a regulatory protein, such as a
transcription factor or DNA binding protein, a structural protein,
such as a cell surface protein, or the target gene product may be a
nucleic acid such as a ribosome or antisense molecule.
[0046] In a preferred embodiment, the target gene products which
provide enhanced properties to the genetically engineered cells,
include but are not limited to, gene products which enhance cell
growth, e.g., vascular endothelial growth factor (VEGF), hepatocyte
growth factor (HGF), fibroblast growth factors (FGF), platelet
derived growth factor (PDGF), epidermal growth factor (EGF), and
transforming growth factor (TGF). In another preferred embodiment,
the cells and tissues are genetically engineered to express target
gene products which result in cell immortalization, e.g. oncogenes
or telomerese. In yet another embodiment, the cells may be
engineered to express a suicide gene product on cue, e.g.,
thymidine kinase.
[0047] In another preferred embodiment, the cells and tissues are
genetically engineered to express gene products which provide
protective functions in vitro such as cyropreservation and
anti-desiccation properties, e.g., trehalose (U.S. Pat. Nos.
4,891,319; 5,290,765; 5,693,788). The cells and tissues of the
present invention may also be engineered to express gene products
which provide a protective function in vivo, such as those which
would protect the cells from an inflammatory response and protect
against rejection by the host's immune system, such as HLA
epitopes, major histocompatibility epitopes, immunoglobulin and
receptor epitopes, epitopes of cellular adhesion molecules,
cytokines and chemokines.
[0048] There are a number of ways that the target gene products may
be engineered to be expressed by the cells and tissues of the
present invention. The target gene products may be engineered to be
expressed constitutively or in a tissue-specific or
stimuli-specific manner. In accordance with this aspect of the
invention, the nucleotide sequences encoding the target gene
products may be operably linked to promoter elements which are
constitutively active, tissue-specific or induced upon presence of
a specific stimuli.
[0049] In a specific embodiment, the nucleotide sequences encoding
the target gene products are operably linked to regulatory promoter
elements that are responsive to shear or radial stress. In this
instance, the promoter element would be turned on by passing blood
flow (shear) as well as the radial stress that is induced as a
result of the pulsatile flow of blood through the heart or
vessel.
[0050] Examples of other regulatory promoter elements include
tetracycline responsive elements, nicotine responsive elements,
insulin responsive element, glucose responsive elements, interferon
responsive elements, glucocorticoid responsive elements
estrogen/progesterone responsive elements, retinoid acid responsive
elements, viral transactivators, early or late promoter of SV40
adenovirus, the lac system, the trp system, the TAC system, the TRC
system, the promoter for 3-phosphoglycerate and the promoters of
acid phosphatase. In addition, artificial response elements could
be constructed, composed of multimers of transcription factor
binding sites and hormone-response elements similar to the
molecular architecture of naturally-occurring promoters and
enhancers (e.g., see Herr, W & Clarke, J Cell (1986) 45(3):
461-70). Such artificial composite regulatory regions could be
designed to respond to any desirable signal and be expressed in
particular cell-types depending on the promoter/enhancer binding
sites selected.
5.3 Uses of a Three-Dimensional Stromal Tissue in Promoting
Angiogenesis
[0051] The three-dimensional stromal tissue of the present
invention may be used in a variety of applications including, but
not limited to, promoting repair of and regeneration of damaged
cardiac muscle, promoting vascularization and healing during
cardiac surgery (e.g. by-pass surgery or heart valve replacement),
promoting blood vessel formation at anastomosis sites, and
promoting vascularization and repair of ischemic or otherwise
damaged smooth muscle, cardiac muscle, skeletal muscle, connective
tissue or brain tissue. In that connection, stromal tissue may be
used as a freshly cultured tissue, as a cryopreserved tissue, or
even as a killed tissue.
[0052] The three-dimensional stromal tissue of the present
invention may be attached to various locations on the heart,
including the epicardium, myocardium and endocardium, to promote
angiogenesis in the region of attachment. Means for attachment
include, but are not limited to, direct adherence between the
stromal tissue and the heart tissue, biological glue, synthetic
glue, laser dyes, or hydrogel. A number of commercially available
hemostatic agents and sealants include "SURGICAL" (oxidized
cellulose), "ACTIFOAM" (collagen), "FIBRX" (light-activated fibrin
sealant), "BOHEAL" (fibrin sealant), "FIBROCAPS" (dry powder fibrin
sealant), polysaccharide polymers p-GlcNAc ("SYVEC" patch; Marine
Polymer Technologies), Polymer 27CK (Protein Polymer Tech.).
Medical devices and apparatus for preparing autologous fibrin
sealants from 120 ml of a patient's blood in the operating room in
one and one-half hour are also known (e.g. Vivostat System).
[0053] In an embodiment of the invention utilizing direct
adherence, the three-dimensional stromal tissue is placed directly
onto the heart or an adjoining vessel and the product attaches via
natural cellular attachment. This method has been demonstrated in
studies of wound healing in patients with diabetic foot ulcers.
[0054] In a preferred embodiment, a three-dimensional stromal
tissue is attached to the heart or adjoining vessel using a
surgical glue, preferably a biological glue such as a fibrin glue.
The use of fibrin glue as a surgical adhesive is well known. Fibrin
glue compositions are known (e.g., see U.S. Pat. Nos. 4,414,971;
4,627,879 and 5,290,552) and the derived fibrin may be autologous
(e.g., see U.S. Pat. No. 5,643,192). The glue compositions may also
include additional components, such as liposomes containing one or
more agent or drug (e.g., see U.S. Pat. Nos. 4,359,049 and
5,605,541) and include via injection (e.g., see U.S. Pat. No.
4,874,368) or by spraying (e.g., see U.S. Pat. Nos. 5,368,563 and
5,759,171). Kits are also available for applying fibrin glue
compositions (e.g., see U.S. Pat. No. 5,318,524).
[0055] In another embodiment, a laser dye is applied to the heart
and/or vessel wall, the three-dimensional stromal tissue, or both,
and activated using a laser of the appropriate wavelength to adhere
to the tissues. In preferred embodiments, the laser dye has an
activation frequency in a range that does not alter tissue function
or integrity. For instance, 800 nm light passes through tissues and
red blood cells. Using indocyan green (ICG) as the laser dye, laser
wavelengths that pass through tissue may be used. A solution of 5
mg/ml of ICG is painted onto the surface of the three-dimensional
stromal tissue (or target site) and the ICG binds to the collagen
of the tissue. A 5 ms pulse from a laser emitting light with a peak
intensity near 800 nm is used to activate the laser dye, resulting
in the denaturation of collagen which fuses elastin of the adjacent
tissue to the modified surface.
[0056] In another embodiment, the three-dimensional stromal tissue
is attached to the heart or vessel using a hydrogel. A number of
natural and synthetic polymeric materials are sufficient for
forming suitable hydrogel compositions. For example,
polysaccharides, e.g., alginate, may be crosslinked with divalent
cations, polyphosphazenes and polyacrylates are crosslinked
ionically or by ultraviolet polymerization (U.S. Pat. No.
5,709,854). Alternatively, a synthetic surgical glue such as
2-octyl cyanoacrylate ("DERMABOND", Ethicon, Inc., Somerville,
N.J.) may be used to attach the three-dimensional stromal
tissue.
[0057] In an alternative embodiment of the present invention, the
three-dimensional stromal tissue is secured to the heart or a blood
vessel vessels using one or more sutures, including, but not
limited to, 5-0, 6-0 and 7-0 proline sutures (Ethicon Cat. Nos.
8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyglactin
or other suitable non-biodegradable or biodegradable suture
material. When suturing, double armed needles are typically,
although not necessarily, used.
[0058] In another embodiment, the three-dimensional stromal tissue
is grown in a bioreactor system (e.g., U.S. Pat. Nos. 5,763,267 and
5,843,766) in which the framework is slightly larger than the final
tissue-engineered product. The final product contains a border, one
edge, flap or tab of the scaffold material, which is used as the
site for application of the biological/synthetic glue, laser dye or
hydrogel. In alternative embodiments, the scaffold weave may be
used as an attachment for suturing or microsuturing.
[0059] The three-dimensional stromal tissue may be implanted to
promote vascularization, repair and regeneration of damaged cardiac
muscle. In a preferred embodiment, the three-dimensional stromal
tissues will be applied to a vessel to sprout new blood vessels to
by-pass clogged or blocked arteries and restore blood flow to the
heart. In another embodiment, the three-dimensional stromal tissue
will be applied directly to the heart using a minimally invasive
procedure. The tissue can be applied to promote vascularization and
blood flow to minimize necrosis and/or promote regeneration of
heart tissue following a myocardial infarction. When attaching a
three-dimensional stromal tissue to the heart epicardium or
myocardium, it will be necessary to open the pericardium (i.e., the
heart sac) prior to application. However, attaching a
three-dimensional stromal tissue patch to the endocardium may be
accomplished by inserting a catheter or similar device into a
ventricle of the heart and adhering or attaching the stromal patch
to the wall of the ventricle. It is preferred that the site of
attachment should have a reasonably good blood flow to support
angiogenesis.
[0060] The angiogenic activity of the three-dimensional stromal
tissues may also be used for treating anastomoses. An anastomosis
is defined as an operative union between two hollow or tubular
structures or an opening created by surgery, trauma or disease
between two or more separate spaces or organs (see, e.g., Stedman's
Medical Dictionary, 26.sup.th Ed, Williams & Wilkins,
Baltimore, Md.). For instance, anastomotic sites arise from the
introduction of a vascular graft during a coronary artery bypass
graft (CABG) procedure, during a bowel resection or organ
transplant. In CABG procedures, a three-dimensional tissue is
placed at the site of downstream attachment of the bypass graft to
promote angiogenesis upon restoration of blood flow to that site,
i.e. to form additional arteries arising from the connection sites
in addition to promoting healing of the site. Examples in the
vascular field include, but are not limited to, precapillary
(between arterioles), Riolan's (marginal artery of the colon
connecting the middle and left colic arteries), portal-systemic
(superior-middle/inferior rectal veins; portal vein-inferior vena
cava), termino-terminal (artery to vein) and cavo-pulmonary
(treating cyanotic heart disease by anastomosing the right
pulmonary artery to the superior vena cava).
[0061] In one embodiment, the three-dimensional stromal tissue is
wrapped around the anastomotic site to promote healing of the site
(i.e., endothelialization). In another embodiment, the cells of the
three-dimensional stromal tissue are killed (e.g., by freezing and
thawing) and the resulting product is applied to the site (i.e.,
"TRANSCYTE").
[0062] As described above, encompassed within the scope of the
invention is a method for treating ischemic damage in tissues
including, but not limited to, heart, brain peripheral tissues and
visceral organs. A three-dimensional stromal tissue implant is
attached to the ischemic site using natural adherence, a suture,
adhesive or other means as described above. The implanted
three-dimensional stromal tissue promotes formation of new blood
vessels and healing of the damaged tissue.
[0063] Also encompassed within the scope of the invention is a kit
for promoting angiogenesis comprising a three-dimensional stromal
tissue and a means for attaching such tissue to the heart or
vessels. Such means for attachment include a composition of
surgical glue, hydrogel, preloaded prolene needles for
microsuturing.
6. EXAMPLE: THREE-DIMENSIONAL STROMAL TISSUE PROMOTED
ANGIOGENESIS
[0064] This section demonstrates that a fibroblast-based
three-dimensional stromal tissue ("stromal tissue") was capable of
inducing endothelialization and vascularization. Providing, such a
biologically active material has been observed to induce new
capillary formation and reduce inflammation in the wound bed of
patients with diabetic foot ulcers.
[0065] The angiogenic properties of three-dimensional stromal
tissues are described below using a wide range of techniques
including the chick chorioallantoic membrane assay, the rat aortic
ring assay, stimulation of endothelial cell proliferation,
chemokinesis, chemotaxis, inhibition of apoptosis, and in vivo
induction of angiogenesis in ischemic heart tissue. Collectively,
these assays cover a wide range of the individual events in
angiogenesis as well as the overall process.
[0066] The fibronectin present in the extracellular matrix also has
been shown to stimulate the proliferation of endothelial cells,
while the denatured collagen has been proven to be a favorable
substrate for human endothelial cell attachment. Bound growth
factors in the matrix include TGF.beta. and HGF which are important
in stimulating new capillary formation and endothelialization. The
matrix also contains laminin-1 which can serve to inhibit initial
hyperplasia via the YIGSR peptide. The combination of these matrix
proteins along with naturally secreted growth factors offers a
physiological solution to the in vivo induction of
angiogenesis.
6.1 Materials and Methods
6.1.1. Expression of Growth Factors by Three-Dimensional Stromal
Tissue
[0067] Experiments were performed to examine the expression of
angiogenic factors by the stromal tissues. Growth factor expression
was examined both by estimation of mRNA by polymerase chain
reaction (PCR) methods and estimation of the free protein by
enzyme-linked immunosorption assay (ELISA).
[0068] Specific messenger RNAs were estimated by quantitative
RT-PCR using the ABI TaqMan method (Perkin-Elmer, Foster City,
Calif.). RNA was extracted from the cells using a Rapid RNA
Purification Kit (Amresco, Solon, Ohio). The RNA was reverse
transcribed using Superscript II (Life Technologies, Grand Island,
N.Y.) with random hexamer primers (Sigma, St. Louis, Mo.).
Amplification of samples of cDNA containing 200 ng total RNA was
detected in real time and compared with the amplification of
plasmid-derived standards for specific mRNA sequences using a copy
number over a range of 5 orders of magnitude with
40-4,000,000/reaction. In purification and the efficiency of
reverse transcription, mRNA sequences for PDGF B chain, VEGF or
TGF.beta.1 were added to RNA isolations, and their yield measured
by the TaqMan procedure. The control mRNA sequences were obtained
by T7 RNA polymerase transcription of plasmids containing the
corresponding sequence. The values were normalized using
glyceraldehyde-3-phosphate dehydrogenase as a control.
6.1.2. Chick Chorioallantoic Membrane Assay
[0069] Ten day old chicken embryos were obtained from McIntyre
Farms (Lake, Calif.) and incubated at 37.degree. C. Eggs were
candled to locate and mark a target area void of large vessels. Two
small holes were made in the shell with a needle, directly over the
air sac and over the target area. Suction was applied to the first
hole, causing the CAM to drop away from the marked area. Using a
"DREMEL MOTO-TOOL", the egg shell was removed from the target area
to create a "window." A 4 mm diameter circular sample
(three-dimensional stromal tissue or control) was then placed on
the membrane near, but not on top of, a large blood vessel. The
hole was covered with a piece of clear adhesive tape and the eggs
were incubated for 72 hours at 37.degree. C. to allow blood vessel
growth. The treated section of the membrane was then removed,
photographed, and fixed in methanol. The number of fine blood
vessel branch points in the region of the sample was counted.
Biopsy samples were fixed in methanol and sections stained with
Masson's Trichrome.
6.1.3. Aortic Ring Assay
[0070] In the aortic ring assay, the ability of the endothelial
blood vessel lining to generate microvessels was used to
demonstrate angiogenesis. Thoracic aortas removed from 1 to 2 month
old Sprague Dawley male rats were transferred to serum-free
MCDB131. The peri-aortic fibroadipose tissue was carefully removed,
the aortas washed 8 to 10 times and cut into 1 mm lengths. Wells
were punched in a 1.5% agarose gel and filled with clotting
fibrinogen solution (20 .mu.L 50 NIH units/mL bovine thrombin in 1
mL fibrinogen). The aortic rings were placed into the centers of
the wells. After clotting, the dishes were flooded with serum-free
MCDB131. The cultures were incubated at 37.degree. C. with 5% CO,
with medium changes every 3 days. Newly formed microvessels were
counted on days 3, 7 and 14.
6.1.4. Endothelial Cell Proliferation Assay
[0071] Endothelial cell proliferation is a critical component of
angiogenesis. The ability of the stromal tissue to stimulate this
activity was determined by [.sup.3H]-thymidine incorporation.
Various growth factors and concentrated conditioned medium samples
were assessed for their influence on the proliferation of HUVEC.
Confluent cultures were detached and re-suspended in HUVEC growth
medium to a final concentration of 2.5.times.10.sup.4 cells/ml.
24-well plates were pre-treated with Attachment Factor Solution
(Cell Applications, Inc.) and cells were added at 1 ml cell
suspension per well. Cells were allowed to settle and attach, and
then were switched to Endothelial Serum Free Medium (Cell
Applications, Inc.), supplemented with fibroblast culture medium or
medium conditioned by monolayer or three-dimensional fibroblast
cultures. On day two, the cells received fresh serum free medium
supplemented as appropriate with 1 .mu.Curie/ml
[.sup.3H]-thymidine. On day three, medium was removed, cells were
washed three times with PBS, and 250 .mu.l 2.3% sodium dodecyl
sulfate (SDS) was added to solubilize the cells. After 30 minutes;
the SDS extract and one ml of a PBS wash were transferred to a
scintillation vial. Five ml of "SCINTIVERSE" (Fair Lawn, N.J.) was
added to vials and radioactivity was determined using a Beckman
LS6500 Scintillation Counter (Fullerton, Calif.).
6.1.5. Endothelial Cell Chemokinesis Assay
[0072] The ability of our three-dimensional stromal tissue to
stimulate endothelial cell migration was tested in two ways. The
first was a chemokinesis assay that determined the stimulation of
cell movement without any directional definition. The second
measured cell migration towards a stimulation source.
[0073] Endothelial cells were grown on Cytodex-2 beads. The assay
estimated the dissociation of cells from the beads and
re-association with a culture plate. The cells on the plate were
stained and counted.
6.1.6. Endothelial Cell Chemotaxis Assay
[0074] Cell migration was analyzed with an endothelial cell
chemotaxis assay utilizing a Neuro Probe 48-well Boyden chemotaxis
chamber (Neuro Probe, Inc.). Polycarbate membrane filters (Poretics
Corporation, 25.times.80 mm) were soaked in 0.5M acetic acid
overnight, washed three times for 1 hour with water, incubated in a
solution of 0.01% calf skin gelatin type III, (Sigma, St. Louis,
Mo.) for 12-16 hours, and air dried. HUVECs were detached and
resuspended in HUVEC growth medium at a final concentration of
1.0.times.10.sup.5 cells/ml. The Boyden Chamber was assembled as
follows: 30 .mu.l of sample or standard was added to the bottom
wells, the gelatin coated membrane was placed on top, and 50 .mu.l
cell suspension was added to the upper wells. The chamber was
incubated at 37.degree. C. for 3 hours. Membranes were then
carefully removed from the chamber and the cell-side was rinsed in
PBS and drawn across a wiper blade to remove non-migrated cells.
The membranes were stained with Wright's Giemsa stain and either
the number of cells counted or the density of staining was reported
against a standard curve generated with 20, 10, 5.0 and 0 ng/ml
purified VEGF.
6.1.7. Induction of Integrin
[0075] The .alpha.v.beta.3 integrin has been shown to play an
important role in angiogenesis and neutralizing antibodies directed
at it are capable of blocking capillary blood vessel formation. It
is induced by VEGF and is thought to play a critical role in the
endothelial cell migration.
[0076] The presence of integrins and cell surface receptors was
determined by flow cytometry on a FACStar by Cytometry Research
Services, San Diego, Calif. Cells were prepared for analysis as
follows: HUVECs were trypsinized and the cells re-suspended at
1.times.10.sup.6 cells/ml. 250 .mu.L to 500 .mu.L of the cell
suspensions were washed three times with Hank's Balanced Salt
Solution (HESS, GibcoBRL, Grand Island, N.Y.), and finally
re-suspended in 10% FBS in Hank's balanced salt solution (HBSS).
The cells were incubated for 30 minutes with primary antibodies
diluted to 1 .mu.g/ml in 10% FBS in HBSS, washed three times with
HBSS, incubated for 30 minutes with secondary antibodies diluted to
1 .mu.g/mL in 10% FBS in HBSS, washed three times with HBSS, and
fixed in 200 .mu.L 10% Formalin (Baxter, Deerfield, Ill.) at a
density of 10' cells/mL.
6.1.8. Inhibition of Endothelial Cell Apoptosis
[0077] It has been previously reported that endothelial cells
cultured as a monolayer on "MATRIGEL", a basement membrane growth
substrate (Collaborative Research) coalesced into tubes and
underwent apoptosis. The inclusion of an angiogenic factor, e.g.,
VEGF in the "MATRIGEL", however, maintained endothelial cell
proliferation and morphology suggesting that the angiogenic
activity of VEGF inhibited apoptosis (e.g., see Goto et al., 1993,
Lab Invest. 69:508-517; and Haralbopoulos et al., 1994, Lab Invest.
71:575-582).
[0078] To further evidence the angiogenic activity of the
three-dimensional stromal tissues of the present invention, growth
medium conditioned by a three-dimensional stromal tissue was added
to endothelial cells cultured on "MATRIGEL" to demonstrate the
inhibition of apoptosis. "MATRIGEL" was thawed and solidified in
"TRANSWELL" (Costar, Boston, Mass.) 6-well tissue culture dishes
according to the manufacturer's instructions. Dermal microvascular
endothelial cells (DMEC) were seeded onto the solidified "MATRIGEL"
at 2.5.times.10.sup.5 cells/well, in the presence of growth medium
conditioned by a monolayer culture of fibroblasts or
three-dimensional fibroblast culture, and incubated at 37.degree.
C. in a 5% CO.sub.2 atmosphere as previously described (e.g., see
Kuzuya et al., 1994, J. Cell Physiol. 161:267-276). The DMEC cells
of each culture were stained by incubating the culture in a
solution of 10 .mu.g/ml di-1-acetyl-low density lipoprotein for 2-4
hours (e.g., see Voyta et al., 1984, J. Cell Biol. 99:2034-2040)
and a solution of "SYTOX", which stained cell nuclei (Molecular
Probes, Eugene, Oreg.).
6.1.9. Blood Flow Changes in Human Diabetic Foot Ulcers
[0079] Cultured three-dimensional stromal tissue provides many of
the components of healthy skin essential for wound healing,
including important mediators of angiogenesis like VEFG and
transforming growth factor-.beta. (TGF.beta.). Laser Doppler
imaging was used to study microvascular perfusion at the base of
foot ulcers treated with three-dimensional stromal tissue, to
investigate whether healing of these lesions was associated with an
increase in blood flow that might in turn reflect angiogenesis.
[0080] Seven full-thickness ulcers were assessed in five patients
with type 2 diabetes mellitus. All lesions had been present for at
least three months with no clinical evidence of infection or change
in size over the previous two weeks, despite conventional
treatment. Three-dimensional stromal tissue was applied weekly to
the base of each wound for a total of eight weeks, after which
conventional treatment was resumed. Microvascular perfusion was
assessed using laser Doppler imaging (Moore Instruments, Axminster,
UK) immediately before and after 2, 5 and 8 weeks of treatment.
6.1.10. Stimulation of Vascularization in a Mouse Epicardial
Implant Model
6.1.10.1. Animals
[0081] Three-dimensional stromal tissue-stimulated vascularization
was examined in vivo using a Severe Combined Immunodeficiency
(SCID) mouse epicardial implant model. Mice were divided into three
groups: viable/cryopreserved three-dimensional stromal tissue
implant ("viable stromal patch"), non-viable three-dimensional
stromal tissue implant ("non-viable stromal patch"), and
control/sham. Each group had at least six animals per group at two
separate time points (14 days and 30 days). The animal study was
performed in accordance with applicable regulations of the United
States Food and Drug Administration.
6.1.10.2. Animal Husbandry
[0082] SCID mice (University of Arizona, Tucson, Ariz.) were housed
2 per cage in micro-isolator cages on wood shavings and received
"TECH-LAD 4% MOUSE/RAT DIET" and tap water ad libitum. Mice were
housed under controlled temperatures of 74.degree. F..+-.10.degree.
F. and humidity 50%.+-.20% in accordance with the NIH "Guide for
the Care and Use of Laboratory Animals".
6.1.10.3. Surgical Procedures
[0083] General anesthesia was induced and maintained by an
intraperitoneal injection of 2.5% Avertin. Sterility was maintained
and a warming pad was used throughout the procedure. Mice were
weighed, and the chest wall shaved and prepared. In the supine
position, a tracheotomy was performed, and mice ventilated using a
small animal respirator (tidal volume=0.5 ml, rate=120-130
breaths/min). Proper intubation was confirmed by observation of
chest expansion and retraction during ventilated breaths.
[0084] All surgical procedures were carried out using an operating
microscope. A left thoracotomy was performed and the pectoralis
muscle groups were cut transversely, exposing the thoracic cage.
The fourth intercostal space was entered using scissors and blunt
dissection. Two 6-0 silk sutures (Ethicon) were placed around the
upper and lower ribs for retraction. The thymus was retracted
upward, and the left lung collapsed using a sterile cotton swab.
Pressure was then applied to the right thorax to displace the heart
in a leftward direction.
[0085] To induce epicardial/myocardial ischemic damage, a coronary
occlusion of the left coronary artery just below the left atrium
was performed by thermal occlusion using standard methods known to
those of skill in the art. Occlusion results in an area of
non-viable, ischemic tissue located primarily in the left ventricle
near the apex. A 4 mm viable stromal patch or non-viable stromal
patch was sutured onto the surface of the ischemic
epicardial/myocardial tissue of surviving mice using a single
suture. For control mice, only a suture was introduced at the site
of ischemic damage. Following implantation, the lungs were
re-expanded using positive pressure at end expiration. The chest
cavity was closed in layers using 6-0 silk (Ethicon, Inc.) and the
animal were gradually weaned from the respirator. Once spontaneous
respiration was resumed, the tracheal tube was removed, and the
neck closed. The animals remained in a supervised setting until
fully conscious and the post-operative general health status of
each animal was determined daily.
[0086] Prior to explant, an echocardiogram was performed to measure
ventricular wall thickness and compare to that prior to occlusion.
At 14 days or 30 days, mice were re-anesthetized and the
three-dimensional stromal tissue patches with surrounding tissue
and control heart tissues were harvested. Mice were euthanized
after material harvest using an overdose (150 mg/kg) of
pentobarbital IP.
6.1.10.4 Analyses
[0087] The in vivo formation of new blood vessels in stromal
patch-treated animals and controls was examined using three
separate analyses: gross morphology, histology and
histochemistry.
[0088] The gross morphology of a representative heart from each
group was examined to access the tissue viability in the ischemic
region. The gross morphology of the heart was examined by injecting
one explanted heart from each group with the dye tetrazolium red
(2,3,5-triphenyltetrazolium chloride) (Sigma/Aldrich Chemical Co.,
St. Louis, Mo.). Tetrazolium red reacts with viable heart tissue
producing a bright red color. In contrast, non-viable tissue does
not react with tetrazolium red thus leaving non-viable tissue a
pale white color. Explanted hearts from 14 day and 30 day control
mice and stromal patch-treated mice exhibited a region of
non-viable ischemic heart tissue located primarily in the left
ventricle resulting from the induced coronary occlusion/myocardial
infarction. Images taken at low and high power revealed a large
area of non-viable heart tissue, as evidenced by the pale white
color. In the controls, the ischemic area is devoid of visible
blood vessels.
[0089] The three-dimensional stromal tissue-dependent formation of
new blood vessel was confirmed by histological analysis of sections
of treated and control heart tissues. For histological analysis,
the stromal patch implants and adjacent tissues were excised and
placed in "HISTOCHOICE" fixative (MANUFACTURER) and processed for
light microscopy. The stromal tissue patches and surrounding
tissues were sectioned, placed on slides and stained using
hematoxylin and eosin (H & E). Histological staining using H
& E is well known to those of skill in the art (e.g., In
Histology: A Text and Atlas, 3rd ed. (Ross et al., ed), pp. 1-7;
Williams and Wilkins, Baltimore, Md.) and kits and reagents are
readily available from commercial suppliers (e.g., Sigma/Aldrich
Chemical Co., St Louis, Mo.).
[0090] In addition, the three-dimensional stromal tissue-dependent
formation of new blood vessels was verified using histochemistry to
specifically identify the presence and location of vascular
endothelial cells present in histological sections. The stromal
patch implants and surrounding tissues were sectioned, placed on
slides and histochemically stained using GS-1. GS-1 is a
commercially available lectin that primarily binds to the surface
of endothelial cells (Sigma/Aldrich Chemical Co.).
6.2. Results
6.2.1. Three-Dimensional Stromal Tissue Expressed Angiogenic Growth
Factors
[0091] Engineered three-dimensional stromal tissue secreted a
variety of growth factors, some of which are known to play an
important role in tissue regeneration and angiogenesis. Angiogenic
growth factors expressed by fibroblast-based three-dimensional
stromal tissue are shown in Table II. Cellular concentrations of
mRNA were determined after 24 h recovery from thawing.
TABLE-US-00002 TABLE II THREE-DIMENSIONAL STROMAL TISSUE EXPRESSED
ANGIOGENIC GROWTH FACTORS Potential Expressed, by Secreted, by
importance Growth factor Q-RT-PCR ELISA in wound healing VEGF
8.10.sup.6 copies/.mu.g RNA 700 pg/10.sup.6 Mainly 121 cells/day
amino acid form PDGF A chain 6.10.sup.5 copies/.mu.g RNA Autocrine
environmental sensor PDGF B chain 0 0 Not made IGF-1 5.10.sup.5
copies/.mu.g RNA Co-stimulator of proliferation EGF 3.10.sup.3
copies/.mu.g RNA Negligible HBEGF 2.10.sup.4 copies/.mu.g RNA KGF
7.10.sup.4 copies/.mu.g RNA Probably requires induction by IL-1
TGF.beta..sub.1 6.10.sup.6 copies/.mu.g RNA 300 pg/10.sup.6 Major
product cells/day TGF.beta..sub.3 1.10.sup.4 copies/.mu.g RNA Minor
product HGF 2.10.sup.4 copies/.mu.g RNA 1 ng/10.sup.6 Cells/day
IL-1.alpha. 1.10.sup.4 copies/.mu.g RNA Below Very low output
detection IL-1.beta. 0 Not produced TNF.alpha. 1.10.sup.7
copies/.mu.g RNA Substantial expression TNF.beta. 0 Not expressed
IL-6 7.10.sup.6 copies/.mu.g RNA 500 pg/10.sup.6 Potentially
cells/day important IL-8 1.10.sup.7 copies/.mu.g RNA 25 ng/10.sup.6
Major product cells/day IL-12 0 Not expressed IL-15 0 Not expressed
NGF 0 Not expressed G-CSF 1.10.sup.4 copies/.mu.g RNA 300
pg/10.sup.6 Potentially cells/day important Angiopoietin-1
1.10.sup.4 copies/.mu.g RNA Probably negligible
6.2.2. Three-Dimensional Stromal Tissue Stimulated Angiogenesis in
the Chick Chorioallantoic Membrane and Rat Aortas
[0092] The three-dimensional FBET induced vessel development in the
CAM to a greater extent as compared to control (FIG. 1A-1D),
including both fine capillary development and evidence for
increased permeability. The development of capillary blood vessels
in CAN treated with FBET was also clearly visible by histology.
This type of capillary development is characteristic of
VEGF-induced angiogenesis. It differed from what was seen with
basic FGF stimulation where the vessels showed a larger diameter
with little or no increase in permeability. When the number of
vessels per sample in the CAM was counted, there was a
statistically significant difference between the effects of
scaffold alone and three-dimensional FBET (FIG. 2). The angiogenic
activity of the three-dimensional tissue was reduced by >90% by
pre-incubation with anti-VEGF neutralizing antibody prior to
placement on the CAM, indicating that VEGF production by FBET was
important in its angiogenic activities. When aortic rings of rat
thoracic aortas were co-cultured with FEET, there was a significant
increase in the number of microvessels formed (FIG. 3). It is
believed that the FEET produces a combination of angiogenic factors
in naturally-secreted ratios that may have a synergistic
effect.
6.2.3. Three-Dimensional Stromal Tissue Stimulated Endothelial Cell
Proliferation
[0093] Three-dimensional stromal tissue-conditioned medium
stimulated human endothelial cell proliferation, as measured by
[.sup.3H]-thymidine incorporation as described in section 6.1.4
above (FIG. 4). The stimulatory activities of the medium were dose
dependent.
6.2.4. Three-Dimensional Stromal Tissue Stimulated Endothelial Cell
Chemokinesis
[0094] As shown in FIG. 5, co-culture of endothelial cells with
three-dimensional stromal tissue induced a marked increase in the
transfer of cells from beads to plate (p=0.0003), which was used as
an indication of chemokinesis. This stimulating activity of the
three-dimensional stromal tissue was inhibited about 60% by
anti-hepatocyte growth factor (HGF) neutralizing antibody,
indicating that HGF was also involved.
6.2.5. Three-Dimensional Stromal Tissue Stimulated Endothelial Cell
Chemotaxis
[0095] Medium conditioned with three-dimensional stromal tissue
also stimulated cell migration in a dose-dependent manner (FIG. 6).
In fact, the stromal tissue stimulated greater chemotaxis as
compared with VEGF, even at 50 ng/L. Anti-VEGF antibody inhibited
cell migration stimulated by stromal tissue conditioned medium by
50%.
6.2.6. Three-Dimensional Stromal Tissue Induced Integrin
.alpha.v.beta.3 Expression
[0096] The presence of .alpha.v.beta.3 integrin on the surface of
endothelial cells was analyzed by flow cytometry after treatment
with medium conditioned by three-dimensional stromal tissue.
Cultured HUVECs displayed substantial surface expression of
.alpha.v.beta.3 integrin under normal culture conditions. However,
medium conditioned by stromal tissue stimulated a significant
increase in expression of this integrin (FIG. 7).
6.2.7. Three-Dimensional Stromal Tissue Inhibited Apoptosis of
Human Dermal Microvascular Cells
[0097] Human dermal microvascular cells, when placed under certain
specific conditions such as in a collagen gel overlay or on
"MATRIGEL", form tubules. The tubules are, however, unstable and
degenerate by apoptosis of the cells within about 3 days. However,
if co-cultured with three-dimensional stromal tissue, microvascular
endothelial cells on "MATRIGEL" continued to proliferate and
apoptosis was inhibited (FIGS. 8A and 8B).
[0098] The conditioned medium obtained from monolayer culture
dermal fibroblasts showed no inhibition of apoptosis with the cells
forming tubules and'undergoing apoptosis (FIG. 8A). In contrast,
the conditioned medium obtained from three-dimensional fibroblast
cultures maintained cellular proliferation and morphology similar
to that observed for angiogenic factors such as bFGF and VEGF (FIG.
8B). The results demonstrate two features: 1) the conditioned
medium of three-dimensional cultures was capable of inhibiting
cellular apoptosis in the "MATRIGEL" assay similar to addition of
angiogenic factors; three-dimensional stromal tissue produced and
secreted VEGF and HGF; and, thus the inhibition of apoptosis is
presumed to be the result of the angiogenic factors secreted into
the medium; and 2) the same fibroblasts grown in monolayer did not
produce such an effect demonstrating that the three-dimensional
culture conditions were responsible for the activity (i.e.
angiogenic factor expression/secretion was nonexistent or greatly
diminished with a monolayer of fibroblasts).
6.2.7. Three-Dimensional Stromal Tissue Stimulated Vascularization
and Increased Blood Flow in Human Diabetic Foot Ulcers
[0099] Blood flow at the base of diabetic foot ulcers treated with
three-dimensional stromal tissue increased significantly over the
eight weeks of treatment, from 325+184 (mean.+-.SD) to a peak of
560+344 arbitrary perfusion units (p<0.001, repeated measures
ANOVA). Five of the lesions had healed by twelve weeks and the
other two had markedly reduced in size. These changes in blood flow
indicate angiogenesis in the newly forming granulation tissue,
enhanced by a sustained and appropriate supply of angiogenic growth
factors provided by the three-dimensional stromal tissue.
[0100] Similarly, photomicrographs taken before and after treatment
with stromal tissue showed rapid vascularization of the wound bed,
remodeling of the wounded tissue, and reduction in inflammation
following treatment (FIGS. 9A-9D).
6.2.8. Three-Dimensional Stromal Tissue Stimulated Vascularization
in Ischemic Heart Tissue
[0101] The in vivo formation of new blood vessels in stromal patch
treated mice and controls was examined using three types of
analyses (gross morphology, histology and histochemistry) as
described in section 6.1.10 above.
6.2.8.1 Gross Morphology and Pathology Results
[0102] With respect to the implanted animals, data obtained from 14
and 30 day stromal patch implanted hearts demonstrated that viable
and non-viable stromal patch implants were well incorporated into
the native heart tissue at the site of implantation. Moreover, the
application of a viable stromal patch at the ischemic site resulted
in the visually observable formation of a number of new blood
vessels in the ischemic area that was not observed in untreated
control animals. For instance, images taken under magnification
clearly demonstrate the presence of numerous blood vessels in the
area of implantation using a viable stromal patch implant. New
blood vessel formation at the area of implantation was also
observed in the non-viable stromal patch hearts. The number of new
blood vessels formed, however, appeared to be appreciably greater
in the viable stromal patch treated mice than non-viable stromal
patch treated animals.
[0103] The gross morphological observations demonstrate that a
three-dimensional stromal tissue of the instant invention is
capable of promoting angiogenesis in heart tissue.
6.2.8.2 Histology Results
[0104] Light micrographs of sections obtained from normal,
untreated SCID mouse hearts illustrate the organization of the
myocardium and the outer most portion of the heart's surface, the
epicardium. The myocardial layer contains arterioles, capillaries
and venules. Compared to normal SCID mice, the induction of
myocardial infarction by coronary occlusion resulted in a dramatic
decrease in the number of detectable venules present in the
epicardial layer.
[0105] In contrast, light micrographs of sections obtained from
stromal patch treated hearts showed numerous new vessels formed in
the epicardial layer and the presence of arterioles located in the
myocardium near the epicardial/myocardial interface. Similarly,
non-viable stromal patch treated animals showed the presence of new
vessel formation in the epicardial layer but to a much lesser
degree than viable stromal patch treated hearts. The histological
results confirm the gross morphological observations that the
three-dimensional stromal tissues of the instant invention promote
new, blood vessel formation.
6.2.8.3 Histochemistry Results
[0106] Light micrographs of sections of stromal patch treated
hearts revealed the presence of vascular endothelial cells lining
vessels in the epicardium as well as venules and arterioles in
myocardium. A reduced number of microvascularization was detected
in non-viable stromal patch treated hearts. In contrast, little
staining was observed of endothelial lined vessels in the
epicardium of control hearts. These results demonstrate that
three-dimensional stromal tissues of the instant invention
stimulate angiogenesis in vivo.
[0107] The present invention is not to be limited in scope by the
exemplified embodiments, which are intended as illustrations of
individual aspects of the invention. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
[0108] All publications cited herein are incorporated by reference
in their entirety.
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