U.S. patent application number 15/963750 was filed with the patent office on 2018-08-30 for anti-thrombogenic grafts.
The applicant listed for this patent is YALE UNIVERSITY. Invention is credited to Sashka DIMITRIEVSKA, Laura NIKLASON.
Application Number | 20180243482 15/963750 |
Document ID | / |
Family ID | 51934323 |
Filed Date | 2018-08-30 |
United States Patent
Application |
20180243482 |
Kind Code |
A1 |
DIMITRIEVSKA; Sashka ; et
al. |
August 30, 2018 |
ANTI-THROMBOGENIC GRAFTS
Abstract
The present invention provides anti-thrombogenic compositions,
including anti-thrombogenic vascular grafts. In certain
embodiments, the compositions comprise decellularized tissue coated
with an anti-thrombogenic coating. The present invention also
provides methods of preparing anti-thrombogenic compositions and
methods of treatment comprising implanting the anti-thrombogenic
compositions into a subject in need thereof.
Inventors: |
DIMITRIEVSKA; Sashka;
(Branford, CT) ; NIKLASON; Laura; (Greenwich,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YALE UNIVERSITY |
New Haven |
CT |
US |
|
|
Family ID: |
51934323 |
Appl. No.: |
15/963750 |
Filed: |
April 26, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14783897 |
Oct 12, 2015 |
9981066 |
|
|
PCT/US2014/038597 |
May 19, 2014 |
|
|
|
15963750 |
|
|
|
|
61825256 |
May 20, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09D 105/08 20130101;
A61L 33/0029 20130101; C08B 37/0072 20130101; A61L 27/507 20130101;
B05D 7/54 20130101; C09D 105/10 20130101; A61L 2430/40 20130101;
A61K 35/44 20130101; A61L 27/54 20130101; A61L 27/34 20130101; A61L
2300/608 20130101; A61L 33/0064 20130101; A61L 33/08 20130101; A61L
33/0011 20130101; A61L 2420/08 20130101; A61K 31/727 20130101; A61L
2420/02 20130101; A61L 2300/42 20130101; A61L 27/52 20130101; A61L
2300/236 20130101; C08B 37/0075 20130101; A61L 27/3625 20130101;
A61L 27/34 20130101; C08L 5/08 20130101 |
International
Class: |
A61L 27/50 20060101
A61L027/50; A61K 31/727 20060101 A61K031/727; A61L 27/34 20060101
A61L027/34; A61L 27/36 20060101 A61L027/36; A61L 27/52 20060101
A61L027/52; A61L 33/00 20060101 A61L033/00; B05D 7/00 20060101
B05D007/00; A61L 33/08 20060101 A61L033/08; A61L 27/54 20060101
A61L027/54 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
HL083895 awarded by National Institute of Health. The government
has certain rights in the invention.
Claims
1. A composition comprising a substrate having at least one surface
coated with an anti-thrombogenic coating comprising thiol-modified
hyaluronic acid.
2. The composition of claim 1, wherein the anti-thrombogenic
coating is crosslinked to the at least one surface of the
substrate.
3. The composition of claim 1, wherein the anti-thrombogenic
coating comprises a therapeutic agent.
4. The composition of claim 1, wherein the substrate is
decellularized tissue.
5. The composition of claim 4, wherein the decellularized tissue is
a decellularized blood vessel having a luminal surface, and wherein
the anti-thrombogenic coating is coated on the luminal surface of
the decellularized blood vessel.
6. A method of preparing a graft coated with an anti-thrombogenic
coating comprising thiol-modified hyaluronic acid, comprising the
steps of: providing a substrate having at least one surface; and
coating the at least one surface with an anti-thrombogenic coating,
wherein said step of coating comprises: applying a first
crosslinking solution to the surface; and applying a hydrogel
solution to the surface, thereby providing an anti-thrombogenic
coating on the surface of the substrate, wherein the hydrogel
solution comprises thiol-modified hyaluronic acid.
7. The method of claim 6, wherein the substrate is decellularized
tissue.
8. The method of claim 7, wherein the decellularized tissue is a
decellularized blood vessel.
9. A method of treating a diseased blood vessel in a subject,
comprising bypassing the diseased blood vessel by implanting into
the subject an anti-thrombogenic vascular graft, comprising a
substrate having a luminal surface coated with an anti-thrombogenic
coating comprising thiol-modified hyaluronic acid.
10. The method of claim 9, wherein the subject has a disorder
selected from the group consisting of peripheral vascular disease,
atherosclerosis, aneurysm, and venous thrombosis.
11. The method of claim 9, wherein the anti-thrombogenic coating is
crosslinked to the luminal surface of the substrate.
12. The method of claim 9, wherein the substrate is a
decellularized blood vessel.
13. A method of providing vascular access in a subject, comprising
implanting into the subject an anti-thrombogenic vascular graft,
comprising a substrate having a luminal surface coated with an
anti-thrombogenic coating comprising thiol-modified hyaluronic
acid.
14. The method of claim 13, wherein the subject is undergoing or is
anticipated to undergo hemodialysis.
15. The method of claim 13, wherein the anti-thrombogenic coating
is crosslinked to the luminal surface of the substrate.
16. The method of claim 13, wherein the substrate is a
decellularized blood vessel.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S. patent
application Ser. No. 14/783,897, filed Oct. 12, 2015, which is a 35
U.S.C. .sctn. 371 national phase application of, and claims
priority to, International Application No. PCT/US2014/038597, filed
May 19, 2014, and published under PCT Article 21(2) in English,
which claims priority under 35 U.S.C. .sctn. 119(e) to U.S.
Provisional Patent Application No. 61/825,256, filed May 20, 2013,
all of which application s are incorporated herein by reference in
their entireties.
BACKGROUND OF THE INVENTION
[0003] Vascular grafting is the use of transplanted blood vessels
or synthetic scaffolds to replace, repair, or bypass damaged or
potentially dangerous vessels. Vascular grafts are implanted into
subjects with a wide variety of diseases and disorders, including
cardiovascular disease, atherosclerosis, peripheral vascular
disease, abdominal aortic aneurysm, and the like. These grafts can
improve or restore blood flow to regions in which flow is
obstructed. While autologous vessels or synthetic vessels made from
biocompatible materials are traditionally used today, there has
been some development in the use of decellularized structures as
vascular grafts. Decellularized vascular grafts retain the shape
and structure of native vessels, but are devoid of cells, thereby
minimizing the immunogenicity of the grafts. Various decellularized
biological structures are being developed as small-caliber vascular
grafts. Currently their main drawback is high thrombogenicity,
which can be reduced by recellularizing the luminal side of the
implant with host cells. However, this solution implies at least a
one month patient specific waiting time, due to required harvest
and expansion of autologous endothelial cells to line the graft
lumen. For clinical usage of newly emerging biological vascular
grafts (such as tissue engineered and native decellularized grafts)
a solution that will be available at the time of need is necessary
for clinical application.
[0004] Thus, there is a need in the art for anti-thrombogenic
coatings of vascular grafts. The present invention satisfies this
unmet need.
SUMMARY OF THE INVENTION
[0005] As described below, the present invention includes
anti-thrombogenic compositions, such as anti-thrombogenic vascular
grafts, compositions comprising decellularized tissue coated with
an anti-thrombogenic coating, methods of preparing
anti-thrombogenic compositions, and methods of treatment comprising
implanting the anti-thrombogenic compositions into a subject in
need thereof.
[0006] One aspect of the invention includes a composition
comprising a substrate having at least one surface coated with an
anti-thrombogenic coating.
[0007] Another aspect includes a method of preparing a graft coated
with an anti-thrombogenic coating, comprising the steps of:
providing a substrate having at least one surface; and coating the
at least one surface with an anti-thrombogenic coating, wherein
said coating comprises: applying a first crosslinking solution to
the surface; and applying a hydrogel solution to the surface,
thereby providing a first layer on the surface of the
substrate.
[0008] In another aspect, the invention includes a method of
treating a diseased blood vessel in a subject, comprising bypassing
the diseased blood vessel by implanting into the subject an
anti-thrombogenic vascular graft, comprising a substrate having a
luminal surface coated with an anti-thrombogenic coating.
[0009] In still another aspect, the invention includes a method of
providing vascular access in a subject, comprising implanting into
the subject an anti-thrombogenic vascular graft, comprising a
substrate having a luminal surface coated with an anti-thrombogenic
coating.
[0010] In various embodiments of the above aspects or any other
aspect of the invention delineated herein, the anti-thrombogenic
coating comprises a first layer comprising a hydrogel. In one
embodiment, the first layer comprises hyaluronic acid, such as a
thiol-modified hyaluronic acid. In another embodiment, the first
layer is crosslinked to the at least one surface of the substrate,
such as the luminal surface of the substrate. In yet another
embodiment, the hydrogel solution comprises hyaluronic acid.
[0011] In another embodiment, the anti-thrombogenic coating further
comprises a second layer comprising an anti-coagulant, wherein the
second layer is crosslinked to the first layer. In some embodiments
that include a second layer, the second layer comprises heparin. In
another embodiment, the anti-coagulant solution comprises
heparin.
[0012] In yet another embodiment, the substrate is a decellularized
tissue, such as a decellularized blood vessel. In another
embodiment, the decellularized tissue is a decellularized blood
vessel having a luminal surface, and wherein the anti-thrombogenic
coating is coated on the luminal surface of the decellularized
blood vessel.
[0013] In still another embodiment, the coating further comprises
applying a second crosslinking solution to the first layer and
applying an anti-coagulant solution to the first layer, thereby
providing a second layer atop the first layer.
[0014] In still yet another embodiment, the subject has a disorder
selected from the group consisting of peripheral vascular disease,
atherosclerosis, aneurysm, and venous thrombosis. In one
embodiment, the subject is undergoing or is anticipated to undergo
hemodialysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The following detailed description of preferred embodiments
of the invention will be better understood when read in conjunction
with the appended drawings. For the purpose of illustrating the
invention, there are shown in the drawings embodiments which are
presently preferred. It should be understood, however, that the
invention is not limited to the precise arrangements and
instrumentalities of the embodiments shown in the drawings.
[0016] FIG. 1 is a schematic description of HA-heparin based
coating of decellularized grafts structures, using thiolated-HA as
a first layer of coating and "end-on" aminated heparin as a second
layer of the coating.
[0017] FIG. 2A depicts the schematic description of heparin
modification for "end-on" heparin modification and Sulfo-SMCC
addition for spontaneous heparin crosslinking onto hyaluronic acid
coated decellularized vessels.
[0018] FIG. 2B depicts the NMR characterization of heparin
modification.
[0019] FIG. 3 is a set of images depicting the cross-sections of HA
coated decellularized porcine abdominal aortas using increasing
concentrations of SM(PEG).sub.12 crosslinker (NHS-maleimide
crosslinker). As the concentration of the crosslinker increases, so
does the coating smoothness and thickness as demonstrated by the
increasing thick layer of blue dye on the surface (Toluidine Blue),
and orange dye layer (Alamar Blue pH 1). The coating is indicated
by arrows in both Toluidine Blue and Alamar Blue pH 1 (AB pH
1).
[0020] FIG. 4 is a set of images depicting the birds-eye view SEM
images of control decellularized rat abdominal aortas (aortas that
are decellularized with no further treatment), hyaluronic acid
coated decellularized aortas, and layer-by-layer hyaluronic
acid-heparin coated aortas.
[0021] FIG. 5 is an image depicting a SEM cross-section of entire
tubular decellularized rat abdominal aortas layer-by-layer
HA-heparin coated. The coating can be clearly seen on the luminal
side of the vessels as a few microns thick layer as indicated by
white arrow.
[0022] FIG. 6 is a set of images depicting histological sections of
entire tubular control rat abdominal aortas (decellularized aortas
with no further treatment), and layer-by-layer hyaluronic
acid-heparin coated decellularized aortas. The sections were
stained with Toluidine Blue, Alcian Blue pH1, and Alcian Blue PAS.
The coating can be clearly seen on the luminal side of the vessels
as a few microns thick layer.
[0023] FIG. 7 is a set of SEM images of platelets isolated from rat
blood incubated on decellularized control rat abdominal aortas,
hyaluronic acid coated and layer-by-layer hyaluronic acid-heparin
coated decellularized aortas. The platelets and thrombus formation
are clearly visible on the control. The treated vessels show
complete absence of platelets adhesion.
[0024] FIG. 8 is a set of images depicting the results from
experiments where platelets were phalloidin stained (which produces
a red color in the platelets), incubated on decellularized control
rat abdominal aortas, and layer-by-layer hyaluronic acid-heparin
coated decellularized aortas. The platelets are clearly visible on
the control aorta. The coated aortas show an absence of
platelets.
[0025] FIG. 9 is a graph depicting the determination of functional
surface heparin via the Factor X assay where the heparin effects on
Factor X inactivation are expressed in back-calculated heparin
equivalent weights. The assayed samples were: decellularized
control aortas, freshly excised native rat aorta with a continuous
layer of endothelial cells preserved, a cultured continuous
monolayer of HUVECs, and decellularized rat aortas hyaluronic
acid-heparin coated.
[0026] FIG. 10 is an image depicting the results of experiments
wherein HUVECs were plated on HA-heparin layer-by-layer coated
aortas and cultured for 2 weeks. The HUVECs cytoskeleton was
stained with phalloidin (red) and HUVECs nucleus was stained with
DAPI (blue) and imaged over a 10 .mu.m z-stack. The x and y axis
are shown on the sides of the image where the HUVECs are seen
growing a non-planar monolayer. The HUVECs can be seen invading the
coating.
[0027] FIG. 11 is a panel of graphs showing storage modulus (G full
circles) and loss modulus (G'' open circles) of HA gels and HA-PEG
crosslinker incubated onto decellularized porcine aorta plotted as
a function of time. Panels A and B display the Hyaluronic acid
loaded onto decellularized porcine aorta in the absence of the PEG
crosslinker and where the elastic (G') and loss (G'') moduli are
plotted against time in Log (panel A) or Ln (panel B). Panels C and
D display the Hyaluronic acid loaded onto decellularized porcine
aorta with the addition of PEG crosslinker. The elastic (G') and
loss (G'') moduli are plotted against time in Log (panel C) or Ln
(panel D). The storage modulus G was calculated to be 37 kPa for
the HA-PEG and 2 kPa for the HA gels without the PEG crosslinker at
80% polymerized form of the gels using the complex storage modulus
equation above. Of importance to the coating developments, the
HA-PEG gels attain their mature cross-linked form within 4 hours of
incubation and the HA gels alone attain a mature form within 23
hours of incubation. This indicated that after at least 4 hours of
luminal perfusion of the HA gels within the vessels should have
fully polymerized coatings.
[0028] FIG. 12 is a panel of images showing decellularized rat
aortas uncoated control (left-hand panel of images) and
decellularized rat aortas Hyaluronic Acid/ Heparin coated
(right-hand panel of images) stability evaluation at 37.degree. C.
for two weeks incubated under PBS (panel A) and freshly drawn rat
plasma (panel B). Following both PBS and rat plasma incubation the
coating remains present and visible via AB/PAS and Toluidine Blue
stains.
[0029] FIG. 13 is panel of images showing HUVECs seeded onto the
three different coating layers deposited stained with DAPI (blue)
and VE-Cadherin (red) after three days of culture. The coating
components are biocompatible and support endothelial cell growth
and proliferation in vitro on short time periods.
[0030] FIG. 14 is a panel of images showing cross-sections of
rat-decellularized grafts implanted end-to-end in rat abdominal
aortas at after 4 weeks of implantation. The top panel shows an
example of the control group where the implanted graft was a
decellularized rat aorta without further modification. The bottom
panel shown an example of Hylaronic acid coated rat decellularized
graft implanted end-to-end in rat abdominal aorta 4 weeks after
implantation. All the sections were stained with hematoxylin and
eosin (H&E) showing the migration of abluminal cells within the
grafts and the cellular deposition within the large blood clot of
the control group rat.
[0031] FIG. 15 is a panel of images showing explants and Doppler
ultrasound imaging assessment of graft patency at week 4. Top panel
is the control decellularized rat aorta and the bottom panel is the
Hyaluronic Acid coated decellularized rat aorta. Control implants
showed no flow recording as per the Doppler ultrasound imaging
where a flat line is indicative of the absence of flow. The picture
of the explant shown a large fibrotic blood clot well formed in the
center of the implant preventing any blood flow through the graft.
The graft is also dilated to 20.times. its original size which
indicates that the graft wall were probably week and a large
anastomosis formation. On the other hand the Hyaluronic Acid coated
rat decellularized aortas (bottom panel) Doppler recording shows
the typical rat pulsatile flow indicative of healthy vascular flow
and typical rat aorta flow readouts of 30 cm/s velocity peak. The
explant picture shows the absence of luminal occlusions, clots and
blockages and sown that the graft is within the implanted
dimensions of 2 mm diameter indicating the absence of
dilatation.
[0032] FIG. 16 is a panel of images showing cross-sections of
TEVG-decellularized implanted end-to-side in dog carotid arteries
at after 4 weeks of implantation. The top panel shows an example of
the control group where the implanted graft was a decellularized
TEVG without further modification. The bottom panel shown an
example of Hyaluronic acid coated decellularized TEVG. All the
sections were stained with hematoxylin and eosin (H&E). The
absence of blood clots and occlusions is seen in the coated grafts
and the deposition of endothelial cells is also visible on the
Hyaluronic acid-heparin-coated grafts.
DETAILED DESCRIPTION
[0033] The present invention relates to anti-thrombogenic coated
compositions, methods of preparing such compositions, and methods
of using such compositions. For example, in certain embodiments,
the present invention provides vascular grafts coated with an
anti-thrombogenic coating. The present invention is based upon the
discovery that coating the luminal surface of a decellularized
blood vessel with a layer of hyaluronic acid (HA) or a multilayer
coating of HA and heparin or other molecules prevents
thrombogenesis in the vessel. Thus, in one embodiment, the
invention provides an anti-thrombogenic vascular graft composition
comprising a decellularized blood vessel wherein the luminal
surface of the blood vessel is coated with an HA layer. In another
embodiment, the invention provides an anti-thrombogenic vascular
graft composition comprising a decellularized blood vessel wherein
the luminal surface of the blood vessel is coated with a multilayer
coating where at least one layer comprises HA and at least one
other layer comprises heparin. In one embodiment the HA layer is
crosslinked to the luminal surface of the vessel. In one
embodiment, the heparin layer is crosslinked to the HA layer. In
certain embodiments, one or more layers of the coating comprise a
hydrogel. In other embodiments, the HA layer is bound to other
blood contacting surfaces, such as plastics contained in vascular
grafts or catheters, or native vasculature that conducts blood.
[0034] The invention further provides methods of treatment
comprising implanting an anti-thrombogenic composition of the
invention to a recipient. For example, in one embodiment, the
method comprises implanting a HA-coated or HA-heparin-coated
vascular graft into a subject in need thereof. The coated vascular
graft can be used, for example, to treat a subject having a
diseased or blocked blood vessel. In certain embodiments, the
coated vascular graft is used in a method of treating an aneurysm.
In another embodiment, the coated vascular graft is used in a
method of bypassing a diseased or blocked vessel. In another
embodiment, the coated vascular graft is used in a method of
providing vascular access in a subject undergoing or anticipated to
undergo hemodialysis.
Definitions
[0035] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0036] As used herein, each of the following terms has the meaning
associated with it in this section. The articles "a" and "an" are
used herein to refer to one or to more than one (i.e., to at least
one) of the grammatical object of the article. By way of example,
"an element" means one element or more than one element.
[0037] As used herein, the term "abnormal," when used in the
context of organisms, tissues, cells or components thereof, refers
to those organisms, tissues, cells or components thereof that
differ in at least one observable or detectable characteristic
(e.g., age, treatment, or time of day) from those organisms,
tissues, cells or components thereof that display the "normal"
(expected) respective characteristic. Characteristics that are
normal or expected for one cell or tissue type might be abnormal
for a different cell or tissue type.
[0038] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0039] As used herein, to "alleviate" a disease, defect, disorder
or condition means reducing the severity of one or more symptoms of
the disease, defect, disorder or condition.
[0040] As used herein, "anti-coagulant" refers to an agent or class
of agents that prevents coagulation or clotting of blood.
[0041] As used herein, "anti-thrombogenic coating" refers to a
coating that reduces thrombus or blood clot formation.
[0042] As used herein, "autologous" refers to a biological material
derived from the same individual into whom the material will later
be re-introduced.
[0043] As used herein, "allogeneic" refers to a biological material
derived from a genetically different individual of the same species
as the individual into whom the material will be introduced.
[0044] As used here, "biocompatible" refers to any material, which,
when implanted in a mammal, does not provoke an adverse response in
the mammal. A biocompatible material, when introduced into an
individual, is not toxic or injurious to that individual, nor does
it induce immunological rejection of the material in the
mammal.
[0045] As used herein, the terms "biocompatible polymer" and
"biocompatibility" when used in relation to polymers are recognized
in the art. For example, biocompatible polymers include polymers
that are generally neither toxic to the host, nor degrade (if the
polymer degrades) at a rate that produces monomeric or oligomeric
subunits or other byproducts at toxic concentrations in the host.
In one embodiment, biodegradation generally involves degradation of
the polymer in a host, e.g., into its monomeric subunits, which may
be known to be effectively non-toxic. Intermediate oligomeric
products resulting from such degradation may have different
toxicological properties, however, or biodegradation may involve
oxidation or other biochemical reactions that generate molecules
other than monomeric subunits of the polymer. Consequently, in one
embodiment, toxicology of a biodegradable polymer intended for in
vivo use, such as implantation or injection into a patient, may be
determined after one or more toxicity analyses. It is not necessary
that any subject composition have a purity of 100% to be deemed
biocompatible; indeed, it is only necessary that the subject
compositions be biocompatible as set forth above. Hence, a subject
composition may comprise polymers comprising 99%, 98%, 97%, 96%,
95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers,
e.g., including polymers and other materials and excipients
described herein, and still be biocompatible.
[0046] The term "biologically compatible carrier" or "biologically
compatible medium" refers to reagents, cells, compounds, materials,
compositions, and/or dosage formulations which are suitable for use
in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other
complication commensurate with a reasonable benefit/risk ratio.
[0047] As used herein, "coating" refers to a covering that is
applied to the surface of an object, usually a substrate. The
coating may be continuous or non-continuous over the surface of the
substrate. The coating may have one or more layers.
[0048] By "crosslinking" is meant creating a bond that links one
polymer chain to another. Crosslinking may be induced through a
crosslinking agent, solution or source or may be induced through
self-assembly.
[0049] By "crosslinking agent" or "crosslinking source" is meant an
agent that is capable of forming a chemical or ionic links between
molecules. Non-limiting examples of crosslinking agents or sources
include calcium chloride; ammonium persulfate (APS) and
tetramethylethylenediamine (TEMED), glutaraldehyde, epoxides,
oxidized dextran, p-azidobenzoyl hydrazide,
N-[.alpha.-maleimidoacetoxy]succinimide ester, p-azidophenyl
glyoxal monohydrate,
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide,
bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,
disuccinimidyl suberate,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
N-hydroxysuccinimide (NHS), riboflavin, heat, visible light
irradiation, ultraviolet irradiation, blue light irradiation, and
combinations thereof.
[0050] By "crosslinking solution" is meant a crosslinking agent in
a solution or solvent.
[0051] The term "decellularized" or "decellularization" as used
herein refers to a biostructure (e.g., an organ, or part of an
organ, or a tissue), from which the cellular content has been
removed leaving behind an intact acellular infra-structure. Some
organs are composed of various specialized tissues. The specialized
tissue structures of an organ, or parenchyma, provide the specific
function associated with the organ. The supporting fibrous network
of the organ is the stroma. Most organs have a stromal framework
composed of unspecialized connecting tissue which supports the
specialized tissue. The process of decellularization removes the
specialized tissue cells, leaving behind the complex
three-dimensional network of extracellular matrix. The connective
tissue infra-structure is primarily composed of collagen. The
decellularized structure provides a biocompatible substrate onto
which different cell populations can be infused. Decellularized
biostructures can be rigid, or semi-rigid, having an ability to
alter their shapes.
[0052] The term "derived from" is used herein to mean to originate
from a specified source.
[0053] As used herein, "extracellular matrix composition" includes
both soluble and non-soluble fractions or any portion thereof. The
non-soluble fraction includes those secreted ECM proteins and
biological components that are deposited on the support or
scaffold. The soluble fraction includes refers to culture media in
which cells have been cultured and into which the cells have
secreted active agent(s) and includes those proteins and biological
components not deposited on the scaffold. Both fractions may be
collected, and optionally further processed, and used individually
or in combination in a variety of applications as described
herein.
[0054] As used herein, the term "gel" refers to a three-dimensional
polymeric structure that itself is insoluble in a particular liquid
but which is capable of absorbing and retaining large quantities of
the liquid to form a stable, often soft and pliable, but always to
one degree or another shape-retentive, structure. When the liquid
is primarily water, the gel is referred to as a hydrogel.
[0055] As used herein, a "graft" refers to a composition that is
implanted into an individual, typically to replace, correct or
otherwise overcome a cell, tissue, or organ defect. A graft may
comprise a scaffold. In certain embodiments, a graft comprises
decellularized tissue. In some embodiments, the graft may comprise
a cell, tissue, or organ. The graft may consist of cells or tissue
that originate from the same individual; this graft is referred to
herein by the following interchangeable terms: "autograft,"
"autologous transplant," "autologous implant" and "autologous
graft." A graft comprising cells or tissue from a genetically
different individual of the same species is referred to herein by
the following interchangeable terms: "allograft," "allogeneic
transplant," "allogeneic implant" and "allogeneic graft." A graft
from an individual to his identical twin is referred to herein as
an "isograft," a "syngeneic transplant," a "syngeneic implant" or a
"syngeneic graft." A "xenograft," "xenogeneic transplant" or
"xenogeneic implant" refers to a graft from one individual to
another of a different species.
[0056] As used herein, the term "intact" refers to a state of being
whereby an element is capable of performing its original function
to a substantial extent.
[0057] "Photo-crosslinking" refers to bond formation that links one
polymer chain to another upon exposure to light of appropriate
wavelengths. For example, two polymers conjugated to a
photoreactive group can be covalently photo-crosslinked by covalent
bond formation between the photoreactive groups.
[0058] As used herein, the term "polymerization" or "cross-linking"
refers to at least one reaction that consumes at least one
functional group in a monomeric molecule (or monomer), oligomeric
molecule (or oligomer) or polymeric molecule (or polymer), to
create at least one chemical linkage between at least two distinct
molecules (e.g., intermolecular bond), at least one chemical
linkage within the same molecule (e.g., intramolecular bond), or
any combination thereof. A polymerization or cross-linking reaction
may consume between about 0% and about 100% of the at least one
functional group available in the system. In one embodiment,
polymerization or cross-linking of at least one functional group
results in about 100% consumption of the at least one functional
group. In another embodiment, polymerization or cross-linking of at
least one functional group results in less than about 100%
consumption of the at least one functional group.
[0059] As used herein, "scaffold" refers to a structure, comprising
a biocompatible material that provides a surface suitable for
adherence and proliferation of cells. A scaffold may further
provide mechanical stability and support. A scaffold may be in a
particular shape or form so as to influence or delimit a
three-dimensional shape or form assumed by a population of
proliferating cells. Such shapes or forms include, but are not
limited to, films (e.g. a form with two-dimensions substantially
greater than the third dimension), ribbons, cords, sheets, flat
discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.
[0060] As used herein, "substrate" refers to a supporting
material.
[0061] As used herein, "surface" refers to the outer most layer of
a substrate or outermost part of the substrate.
[0062] As used hererin, "thiol-modified" refers to one or more
modifications to the substrate.
[0063] As used herein, to "treat" means reducing the frequency with
which symptoms of a disease, defect, disorder, or adverse
condition, and the like, are experienced by a patient.
[0064] The term "tissue," as used herein includes, but is not
limited to, bone, neural tissue, fibrous connective tissue
including tendons and ligaments, cartilage, dura, pericardia,
muscle, lung, heart valves, veins and arteries and other
vasculature, dermis, adipose tissue, or glandular tissue.
[0065] As used herein, "scaffold" refers to a structure, comprising
a biocompatible material that provides a surface suitable for
adherence of a substance. A scaffold may further provide mechanical
stability and support. A scaffold may be in a particular shape or
form so as to influence or delimit a three-dimensional shape or
form. Such shapes or forms include, but are not limited to, films
(e.g. a form with two-dimensions substantially greater than the
third dimension), ribbons, cords, sheets, flat discs, cylinders,
spheres, 3-dimensional amorphous shapes, etc.
[0066] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, a subject is preferably a mammal
such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats,
etc.) and a primate (e.g., monkey and human), most preferably a
human.
[0067] As used herein, the term "treating a disease or disorder"
means reducing the frequency with which a symptom of the disease or
disorder is experienced by a patient. Disease and disorder are used
interchangeably herein.
[0068] As used herein, the term "therapeutically effective amount"
refers to an amount that is sufficient or effective to prevent or
treat (delay or prevent the onset of, prevent the progression of,
inhibit, decrease or reverse) a disease or condition described or
contemplated herein, including alleviating symptoms of such disease
or condition.
[0069] As used herein, the term "effective amount" or
"therapeutically effective amount" of a compound is that amount of
compound that is sufficient to provide a beneficial effect to the
subject to which the compound is administered.
[0070] Ranges: throughout this disclosure, various aspects of the
invention can be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of
the range.
Description
[0071] The present invention relates to compositions coated with an
anti-thrombogenic coating, methods of making such compositions, and
methods of using such compositions. In particular, the invention
relates to biomaterials, tissue engineered constructs, and the
like, which are coated with an anti-thrombogenic coating.
[0072] For example, in certain embodiments, the present invention
provides vascular grafts coated with an anti-thrombogenic coating.
However, the present invention is not limited to any particular
type of material or construct. Rather, the present invention
encompasses any material or construct coated with the
anti-thrombogenic coating of the invention.
[0073] The present invention is based upon the discovery that
coating the luminal surface of a decellularized blood vessel with a
layer of hyaluronic acid (HA) or a multilayer coating of HA and
heparin or other molecules prevents thrombogenesis in the vessel.
Decellularized tissue has been investigated for use as vascular
grafts. However untreated decellularized grafts are thrombogenic,
unless they are recellularized with endothelial cells to inhibit
clot formation, which is a time intensive process which can limit
their clinical applicability. In one embodiment, the present
invention is directed to a chemical coating, in lieu of cell
seeding, to provide an anti-thrombogenic, anticoagulant graft. In
certain embodiments, the coating is stable under standard
refrigeration, thereby allowing for an off the shelf composition to
be used as needed.
[0074] In one embodiment, the invention provides an
anti-thrombogenic vascular graft composition comprising a
decellularized blood vessel wherein the luminal surface of the
blood vessel is coated with a first layer. In certain embodiments,
the first layer comprises HA. In another embodiment, the invention
provides an anti-thrombogenic vascular graft composition comprising
a decellularized blood vessel wherein the luminal surface of the
blood vessel is coated with a multilayer coating. In certain
embodiments, the multilayer coating comprises a first layer
comprising HA and a second layer comprising heparin. In one
embodiment the HA layer is crosslinked to the luminal surface of
the vessel. In one embodiment, the heparin layer is crosslinked to
the HA layer. In certain embodiments, one or more layers of the
coating comprise a hydrogel.
[0075] In one embodiment, the invention provides a method of making
a composition coated with an anti-thrombogenic coating. In certain
embodiments, the method comprises coating a surface of a substrate
with a first layer. In one embodiment, the substrate is a
decellularized tissue. However, the invention is not limited to any
particular type of substrate. Rather, the method encompasses any
suitable substrate known in the art, including, but not limited to,
native blood vessels, engineered blood vessels, synthetic vascular
grafts made from polymers, and blood-contacting catheters made from
polymers. In one embodiment, the first layer comprises HA. In
certain embodiments, the HA is thiol-modified HA. In certain
embodiments, the method comprises using a crosslinker comprising
N-hydroxysuccinimide ester (NHS) and maleimide to crosslink the
amine groups of the substrate with the sulfhydryl groups of the HA.
In certain embodiments, the method comprises a layer-by-layer
coating procedure. In one embodiment, the method comprises coating
the substrate with a second layer. In certain embodiments, the
second layer is coated atop the first layer. For example, in one
embodiment, the second layer comprises aminated heparin, which is
crosslinked to the HA of the first layer.
[0076] The invention further provides methods of treatment
comprising implanting an anti-thrombogenic composition of the
invention. Such methods include implantation of one or more of a
biomaterial, tissue engineering substrate, artificial organ,
artificial tissue, artificial graft, and the like for treating a
disease, disorder, or tissue defect in a subject in need thereof.
For example, in one embodiment, the method comprises implanting a
HA-coated or HA-heparin-coated vascular graft into a subject in
need thereof. The coated vascular graft can be used, for example,
to treat a subject having a diseased or blocked blood vessel. In
certain embodiments, the coated vascular graft is used in a method
of treating an aneurysm. In another embodiment, the coated vascular
graft is used in a method of bypassing a diseased or blocked
vessel. In another embodiment, the coated vascular graft is used in
a method of providing vascular access in a subject undergoing or
anticipated to undergo hemodialysis.
Composition
[0077] The present invention provides a composition comprising a
surface coated with an anti-thrombogenic coating. In certain
embodiments, the composition comprises a biomaterial, tissue
engineering substrate, artificial organ, or artificial tissue
having at least one surface coated with an anti-thrombogenic
coating.
[0078] In one embodiment, the composition of the invention
comprises a vascular graft having at least one surface coated with
an anti-thrombogenic coating. In one embodiment, the vascular graft
is a tubular vascular graft having an outer surface, an inner or
luminal surface, and a hollow passageway. The tubular vascular
grafts of the invention are biocompatible, properly proportioned as
to appropriate dimensions such as diameter, length and wall
thickness, readily attachable to the intended living tissue such as
by sutures, and offer appropriate handling characteristics such as
good flexibility, bending and resistance to kinking during bending.
In certain embodiments, the tubular vascular graft of the invention
is a conduit through which bodily fluids (e.g., blood) may flow
through. The luminal surface of the tubular vascular graft
therefore is the inner surface of the graft that, when implanted,
is in contact with fluid. These tubular vascular grafts can thus be
used to replace segments of native vessels, or otherwise can be
used as artificial vessels serving to bypass native vessels. In
another embodiment, tubular vascular grafts are used as vascular
access points. In certain embodiments, the tubular vascular graft
of the invention has mechanical properties substantially similar to
native blood vessels. That is, the vascular grafts have the wall
strength to withstand the pressure within the vessel. In one
embodiment, the luminal surface of the tubular vascular graft is
coated with a non-thromobogenic coating. The coating prevents
platelet adhesion and thrombosis.
[0079] In certain embodiments, the tubular vascular graft of the
invention has an inner diameter, outer diameter, length, and wall
thickness as needed to mimic the native vessel being repaired,
replaced, or bypassed. For example, in one embodiment, the tubular
vascular graft of the invention is a small-caliber vascular graft,
having an inner diameter of less than 5cm.
[0080] In one embodiment, the tubular vascular graft of the
invention as an inner diameter of about [1] mm to about [25]
mm.
[0081] In one embodiment, the tubular vascular graft of the
invention as an outer diameter of about [1] mm to about [25]
mm.
[0082] In one embodiment, the tubular vascular graft of the
invention has a wall thickness of about [100] .mu.m to about [2]
mm.
[0083] In one embodiment, the tubular vascular graft of the
invention has a length of about [4] cm to about [100] cm.
[0084] In another embodiment, the vascular graft of the invention
is a sheet graft. The sheet grafts of the invention can be used,
for example, to patch portions of native blood vessels. As such,
the sheet graft comprises a luminal surface that, when administered
to the native vessel, is in contact with the fluid flowing through
the vessel. In one embodiment, the luminal surface of the sheet
graft is coated with a non-thromobogenic coating.
[0085] In certain embodiments, the composition of the invention
comprises a substrate, where the surface comprises at least one
surface coated with a non-thrombogenic coating. The substrate may
be any material or biomaterial known in the art. For example, in
certain embodiments, the substrate is an extracellular matrix
protein composition, a collagen-based composition, an elastin-based
composition, hydrogel, electrospun scaffold, injection molded
polymeric scaffolds, woven and non-woven polymeric scaffolds,
metal-based implants, ceramic composite biomaterials, or other
tissue engineering substrate.
[0086] In one embodiment, the substrate is decellularized tissue.
Decellularized tissue substrates are substrates obtained from
harvesting tissue from a donor source and removing cells and
cellular debris from the harvested tissue. The decellularized
tissue substrates retain the structure of the harvested tissue and
can subsequently be used as tissue engineering substrates to be
implanted into a subject in need. Methods of producing
decellularized tissue substrates are well known in the art.
[0087] The present invention is not limited to any particular type
of decellularized tissue or the manner at which the decellularized
tissue was produced.
[0088] In certain embodiments, decellularization relies on a
chemical methodology. In some instances, decellularization
comprises a chemical methodology combined with mechanical means in
order to remove cells from the tissue. In one aspect, the chemical
solution or otherwise referred to as the decellularization solution
used for decellularization generally includes at least a hypertonic
solution, a detergent, and a chelating agent. In certain
embodiments, the hypertonic solution is a hypertonic sodium
chloride solution. In certain embodiments, the detergent is a
zwitterionic detergent such as CHAPS. In certain embodiments, the
chelating agent is EDTA.
[0089] In one embodiment, the decellularization solution can
include a buffer (e.g., PBS) for osmotic compatibility with the
cells. In some instances, the decellularization solution also can
include enzymes such as, without limitation, one or more
collagenases, one or more dispases, one or more DNases, or a
protease such as trypsin. In some instances, the decellularization
solution also or alternatively can include inhibitors of one or
more enzymes (e.g., protease inhibitors, nuclease inhibitors,
and/or collegenase inhibitors).
[0090] In certain instances, a method of producing a decellularized
tissue substrate includes perfusing the tissue with the
decellularization solution. The pressure for which the
decellularization solution is perfused through the tissue can be
adjusted to the desired pressure. In one embodiment, the
decellularization solution is perfused through the tissue at
perfusion pressure below about 30 mmHg. In one embodiment, the
decellularization solution is perfused through the tissue at
pressures less than about 20 mmHg.
[0091] In certain embodiments, the decellularized tissue substrate
is a decellularized blood vessel. For example, a decellularized
blood vessel can serve as a substrate for tubular vascular grafts
described elsewhere herein. In one embodiment, the
decellularization solution can be introduced into blood vessel to
effect cell removal. In certain embodiments, decellularization of
blood vessels removes the native endothelium lining of the
vessel.
[0092] In one embodiment, the decellularized tissue of the
invention consists essentially of the extracellular matrix (ECM)
component of all or most regions of the tissue. ECM components can
include any or all of the following: fibronectin, fibrillin,
laminin, elastin, members of the collagen family (e.g., collagen I,
III, and IV), glycosaminoglycans, ground substance, reticular
fibers and thrombospondin, which can remain organized as defined
structures such as the basal lamina. Successful decellularization
is defined as the absence of detectable myofilaments, endothelial
cells, smooth muscle cells, epithelial cells, and nuclei in
histologic sections using standard histological staining
procedures. Preferably, but not necessarily, residual cell debris
also has been removed from the decellularized tissue.
[0093] In one embodiment, the decellularization process of a
natural tissue preserves the native 3-dimensional structure of the
tissue. That is, the morphology and the architecture of the tissue,
including ECM components are maintained during and following the
process of decellularization. The morphology and architecture of
the ECM can be examined visually and/or histologically. For
example, the basal lamina on the exterior surface of a solid organ
or within the vasculature of an organ or tissue should not be
removed or significantly damaged due to decellularization. In
addition, the fibrils of the ECM should be similar to or
significantly unchanged from that of an organ or tissue that has
not been decellularized.
[0094] In one embodiment, one or more compounds can be applied in
or on a decellularized tissue to, for example, preserve the
decellularized tissue, or to prepare the decellularized tissue for
recellularization and/or to assist or stimulate cells during the
recellularization process. Such compounds include, but are not
limited to, one or more growth factors (e.g., VEGF, DKK-1, FGF,
BMP-1, BMP-4, SDF-1, IGF, and HGF), immune modulating agents (e.g.,
cytokines, glucocorticoids, IL2R antagonist, leucotriene
antagonists), and/or factors that modify the coagulation cascade
(e.g., aspirin, heparin-binding proteins, and heparin). In
addition, a decellularized organ or tissue can be further treated
with, for example, irradiation (e.g., UV, gamma) to reduce or
eliminate the presence of any type of microorganism remaining on or
in a decellularized tissue.
[0095] Exemplary decellularization methods are used to generate a
decellularized tissue provides a controlled, precise way to destroy
cells of a tissue, while leaving the underlying ECM, including
vascularization, and other gross morphological features of the
original tissue intact. In certain embodiments, the decellularized
substrates are then suitable for seeding with appropriate cells. In
one embodiment, the decellularized substrates are not seeded with
cells. In certain embodiments, the decellularized substrates are
coated with a non-thrombogenic coating described elsewhere herein.
Where the process is performed in vitro, the decellularized tissue
is suitable for implantation into the recipient as a replacement
tissue. The present invention includes methods of fabrication of
engineered tissues built from such substrates.
[0096] Although the source of the tissue is not limited, in
exemplary embodiments, the tissue is from a relatively large animal
or an animal recognized as having a similar anatomy (with regard to
the tissue of interest) as a human, such as a pig, a cow, a horse,
a monkey, or an ape. In some embodiments, the source of the tissue
is human, use of which can reduce the possibility of rejection of
engineered tissues based on the scaffold. In certain embodiments,
the tissue is engineered in vitro from cells, and then subjected to
decellularization. In certain embodiments, the tissue is a blood
vessel obtained from the animal. Any suitable blood vessel may be
used to produce the decellularized blood vessel substrate. For
example, in one embodiment, the decellularized substrate produced
from the aorta, or portion thereof, obtained from the donor animal
or from coronary artery, saphenous vein, posterior tibial artery,
pulmonary artery, external iliac artery, right inferior mammary
artery, radial artery.
[0097] The composition of the invention comprises at least one
surface coated with a non-thrombogenic coating. In certain
embodiments, the non-thrombogenic coating prevents platelet
adhesion and activation, thereby reducing thrombosis. For example,
in certain embodiments, the coating prevents access of collagen, or
other thrombogenic components that may be present in the substrate,
to the blood stream. In one embodiment, the coating of the
invention is a single layer coating. In another embodiment, the
coating of the invention is a multi-layer coating. In one
embodiment, the coating comprises a hydrogel layer. For example, in
certain embodiments, the composition comprises a first layer
comprising a hydrogel crosslinked to the substrate. The first layer
is sometimes referred to herein as the hydrogel layer. In one
embodiment, the hydrogel layer comprises thiol-modified hyaluronic
acid (HA) or dihydrazide-modified HA or un-modified HA.
[0098] The hydrogel may comprise any biopolymer or synthetic
polymer known in the art. For example, the hydrogel may comprise
hyaluronans, chitosans, alginates, collagen, dextran, pectin,
carrageenan, polylysine, gelatin or agarose. (see.: W. E. Hennink
and C. F. van Nostrum, 2002, Adv. Drug Del. Rev. 54, 13-36 and A.
S. Hoffman, 2002, Adv. Drug Del. Rev. 43, 3-12). These materials
consist of high-molecular weight backbone chains made of linear or
branched polysaccharides or polypeptides. Examples of hydrogels
based on synthetic polymers include but are not limited to
(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,
poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO),
PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene),
poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A
copolymers, poly(ethylene imine), etc. (see A. S Hoffman, 2002Adv.
Drug Del. Rev, 43, 3-12).
[0099] Hydrogels can generally absorb a great deal of fluid and, at
equilibrium, typically are composed of 60-90% fluid and only 10-30%
polymer. In a preferred embodiment, the water content of hydrogel
is about 70-80%. Hydrogels are particularly useful due to the
inherent biocompatibility of the cross-linked polymeric network
(Hill-West, et al., 1994, Proc. Natl. Acad. Sci. USA 91:5967-5971).
Hydrogel biocompatibility can be attributed to hydrophilicity and
ability to imbibe large amounts of biological fluids
(Brannon-Peppas. Preparation and Characterization of Cross-linked
Hydrophilic Networks in Absorbent Polymer Technology,
Brannon-Peppas and Harland, Eds. 1990, Elsevier: Amsterdam, pp
45-66; Peppas and Mikos. Preparation Methods and Structure of
Hydrogels in Hydrogels in Medicine and Pharmacy, Peppas, Ed. 1986,
CRC Press: Boca Raton, Fla., pp 1-27). The hydrogels can be
prepared by crosslinking hydrophilic biopolymers or synthetic
polymers. Examples of the hydrogels formed from physical or
chemical crosslinking of hydrophilic biopolymers, include but are
not limited to, hyaluronans, chitosans, alginates, collagen,
dextran, pectin, carrageenan, polylysine, gelatin or agarose. (see:
W. E. Hennink and C. F. van Nostrum, 2002, Adv. Drug Del. Rev. 54,
13-36 and A. S. Hoffman, 2002, Adv. Drug Del. Rev. 43, 3-12). These
materials consist of high-molecular weight backbone chains made of
linear or branched polysaccharides or polypeptides. Examples of
hydrogels based on chemical or physical crosslinking synthetic
polymers include but are not limited to
(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate,
poly(ethylene glycol) (PEO), poly(propylene glycol) (PPO),
PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene),
poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A
copolymers, poly(ethylene imine), etc. (see A. S Hoffman, 2002Adv.
Drug Del. Rev, 43, 3-12). In some embodiments, the transparent
hydrogel scaffold comprises poly(ethylene glycol) diacrylate
(PEGDA).
[0100] Hydrogels closely resemble the natural living extracellular
matrix (Ratner and Hoffman. Synthetic Hydrogels for Biomedical
Applications in Hydrogels for Medical and Related Applications,
Andrade, Ed. 1976, American Chemical Society: Washington, D.C., pp
1-36). Hydrogels can also be made degradable in vivo by
incorporating PLA, PLGA or PGA polymers. Moreover, hydrogels can be
modified with fibronectin, laminin, vitronectin, or, for example,
RGD for surface modification, which can promote cell adhesion and
proliferation (Heungsoo Shin, 2003, Biomaterials 24:4353-4364;
Hwang et al., 2006 Tissue Eng. 12:2695-706). Indeed, altering
molecular weights, block structures, degradable linkages, and
cross-linking modes can influence strength, elasticity, and
degradation properties of the instant hydrogels (Nguyen and West,
2002, Biomaterials 23(22):4307-14; Ifkovits and Burkick, 2007,
Tissue Eng. 13(10):2369-85).
[0101] In certain embodiments, the hydrogel of the invention is
crosslinked. Crosslinking of the hydrogel may be performed using
any suitable method known in the art. In certain embodiments, one
or more multifunctional cross-linking agents may be utilized as
reactive moieties that covalently link biopolymers or synthetic
polymers. Such bifunctional cross-linking agents may include
glutaraldehyde, epoxides (e.g., bis-oxiranes), oxidized dextran,
p-azidobenzoyl hydrazide, N-[.alpha.-maleimidoacetoxy]succinimide
ester, p-azidophenyl glyoxal monohydrate,
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide,
bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,
disuccinimidyl suberate,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
N-hydroxysuccinimide (NHS) and other bifunctional cross-linking
reagents known to those skilled in the art. In certain embodiments,
the hydrogel comprises a photo-activated crosslinking agent. In one
embodiment, one or more components of the hydrogel is cross-linked
upon exposure to UV light.
[0102] In certain embodiments, the hydrogel is crosslinked using a
heterobifunctional crosslinker comprising NHS and maleimide. In a
particular embodiment, the crosslinker links the hydrogel layer
directly to the substrate. For example, in one embodiment, the NHS
reacts with the amine groups on the decellularized vessel
substrate, while the malemide reacts with the sulfhydryl groups on
the thiol-modified HA (FIG. 1).
[0103] In certain embodiments the hydrogel is crosslinked using a
homobifunctional crosslinker comprising imidoester reactive groups
such as the DMA (Dimethyl adipimidate.circle-solid.2 HCl)
crosslinker, which is reactive towards amine groups. In a
particular embodiment, the crosslinker links the hydrogel layer
directly to the substrate. For example, in one embodiment, the
imidoester reacts with the amine groups on the decellularized
vessel substrate, and the amine groups on the dihydrazide-Modified
HA.
[0104] In certain embodiments the hydrogel is crosslinked using
EDC/NHS crosslinker which crosslinks carboxyl and amine groups. In
a particular embodiment, the crosslinker links the hydrogel layer
directly to the substrate. For example, in one embodiment, the
EDC/NHS reacts with the carboxyl groups of the unmodified HA and
the amine groups on the decellularized vessel substrate.
[0105] In certain embodiments, one or more multifunctional
cross-linking agents may be utilized as reactive moieties that
covalently link biopolymers or synthetic polymers. Such
bifunctional cross-linking agents may include glutaraldehyde,
epoxides (e.g., bis-oxiranes), oxidized dextran, p-azidobenzoyl
hydrazide, N-[.alpha.-maleimidoacetoxy]succinimide ester,
p-azidophenyl glyoxal monohydrate,
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide,
bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate,
disuccinimidyl suberate,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC),
N-hydroxysuccinimide (NHS) and other bifunctional cross-linking
reagents known to those skilled in the art.
[0106] In another embodiment utilizing a cross-linking agent,
polyacrylated materials, such as ethoxylated (20) trimethylpropane
triacrylate, may be used as a non-specific photo-activated
cross-linking agent. Components of an exemplary reaction mixture
would include a thermoreversible hydrogel held at 39.degree. C.,
polyacrylate monomers, such as ethoxylated (20) trimethylpropane
triacrylate, a photo-initiator, such as eosin Y, catalytic agents,
such as 1-vinyl-2-pyrrolidinone, and triethanolamine. Continuous
exposure of this reactive mixture to long-wavelength light (>498
nm) would produce a cross-linked hydrogel network
[0107] The stabilized cross-linked hydrogel matrix of the present
invention may be further stabilized and enhanced through the
addition of one or more enhancing agents. By "enhancing agent" or
"stabilizing agent" is intended any compound added to the hydrogel
matrix, in addition to the high molecular weight components, that
enhances the hydrogel matrix by providing further stability or
functional advantages. Suitable enhancing agents, which are admixed
with the high molecular weight components and dispersed within the
hydrogel matrix, include many of the additives described earlier in
connection with the thermoreversible matrix discussed above. The
enhancing agent can include any compound, especially polar
compounds, that, when incorporated into the cross-linked hydrogel
matrix, enhance the hydrogel matrix by providing further stability
or functional advantages.
[0108] Preferred enhancing agents for use with the stabilized
cross-linked hydrogel matrix include polar amino acids, amino acid
analogues, amino acid derivatives, intact collagen, and divalent
cation chelators, such as ethylenediaminetetraacetic acid (EDTA) or
salts thereof. Polar amino acids are intended to include tyrosine,
cysteine, serine, threonine, asparagine, glutamine, aspartic acid,
glutamic acid, arginine, lysine, and histidine. The preferred polar
amino acids are L-cysteine, L-glutamic acid, L-lysine, and
L-arginine. Suitable concentrations of each particular preferred
enhancing agent are the same as noted above in connection with the
thermoreversible hydrogel matrix. Polar amino acids, EDTA, and
mixtures thereof, are preferred enhancing agents. In addition the
gels can be loaded with growth factors: basic fibroblast growth
factor (bFGF) and/or vascular endothelial growth factor (VEGF).
VEGF or bFGF is incorporated to the hyaluronic acid gel prior to
the addition of the crosslinker. Crosslinking then proceeds with no
other modifications entrapping the growth factors within the
hyaluronic acid gels. This promotes re-endothelialization of the
gels by the neighboring endothelial cells of the implantation site.
The growth factors may be added at a concentration of 50
ng/cm.sup.2 area of vessel to be cross-linked. The enhancing agents
can also be added to the matrix composition during the crosslinking
of the high molecular weight components.
[0109] The enhancing agents are particularly important in the
stabilized cross-linked bioactive hydrogel matrix because of the
inherent properties they promote within the matrix. The hydrogel
matrix exhibits an intrinsic bioactivity that will become more
evident through the additional embodiments described hereinafter.
It is believed the intrinsic bioactivity is a function of the
unique stereochemistry of the cross-linked macromolecules in the
presence of the enhancing and strengthening polar amino acids, as
well as other enhancing agents.
[0110] In one embodiment, the hydrogel layer may comprise one or
more therapeutic agents. For example, one or more therapeutic
agents can be embedded within the hydrogel layer. In another
embodiment, the hydrogel layer can be modified with functional
groups for covalently attaching a variety of proteins (e.g.,
collagen) or compounds such as therapeutic agents. Therapeutic
agents include, but are not limited to, analgesics, anesthetics,
antifungals, antibiotics, anti-inflammatories, anthelmintics,
antidotes, antiemetics, antihistamines, antihypertensives,
antimalarials, antimicrobials, antipsychotics, antipyretics,
antiseptics, antiarthritics, antituberculotics, antitussives,
antivirals, cardioactive drugs, cathartics, chemotherapeutic
agents, a colored or fluorescent imaging agent, corticoids (such as
steroids), antidepressants, depressants, diagnostic aids,
diuretics, enzymes, expectorants, hormones, hypnotics, minerals,
nutritional supplements, parasympathomimetics, potassium
supplements, radiation sensitizers, a radioisotope, sedatives,
sulfonamides, stimulants, sympathomimetics, tranquilizers, urinary
anti-infectives, vasoconstrictors, vasodilators, vitamins, xanthine
derivatives, and the like. The therapeutic agent can also be other
small organic molecules, naturally isolated entities or their
analogs, organometallic agents, chelated metals or metal salts,
peptide-based drugs, or peptidic or non-peptidic receptor targeting
or binding agents, or peptide/protein growth factors or cytokines.
It is contemplated that linkage of the therapeutic agent to the
matrix can be via a protease sensitive linker or other
biodegradable linkage. Molecules which can be incorporated into the
hydrogel matrix include, but are not limited to, vitamins and other
nutritional supplements; glycoproteins (e.g., collagen);
fibronectin; peptides and proteins; carbohydrates (both simple
and/or complex); proteoglycans; antigens; oligonucleotides (sense
and/or antisense DNA and/or RNA); antibodies (for example, to
infectious agents, tumors, drugs or hormones); and gene therapy
reagents.
[0111] In certain embodiments, the hydrogel layer, coated along the
luminal surface of the substrate, has a thickness of about [100] nm
to about [3] mm. In certain embodiments, the hydrogel layer is
coated with a second layer. For example, in one embodiment, the
hydrogel layer is coated with a second layer comprising an
anti-coagulant. For example, in one embodiment, the second layer
comprises heparin or derivatives thereof. However, the present
invention is not limited to the use of heparin as an
anti-coagulant. Rather, any known anti-coagulant may be used.
Exemplary anti-coagulants include, but are not limited to vitamin K
antagonists, coumarins, Curcumin (diferuloyl methane), Hirudin,
heparins, Factor Xa inhibitors, direct Xa inhibitors, direct
thrombin inhibitors, natural polysaccharides and synthetic ones
based on .beta.-(1-4) linked anhydroglucose units, chondroitin
sulfate, glycosaminoglycans, and the like.
[0112] In one embodiment, the second layer is crosslinked to the
first layer. For example, in one embodiment, the anti-coagulant of
the second layer is crosslinked to the hyaluronic acid of the first
layer (e.g., hydrogel layer). In one embodiment, the anti-coagulant
of the second layer is modified, which in certain instances allows
for easier crosslinking to the first layer. For example, in certain
embodiments, the second layer comprises aminated heparin, wherein
the heparin comprises a primary amine group. In one embodiment, the
heparin is aminated at the end-chain electrophilic carbon atom
("end-on amination") (FIG. 2).
[0113] In certain embodiments, the aminated heparin is crosslinked
to the hyaluronic acid via EDC/NHS. However, the composition of the
invention is not limited to any particular crosslinker. Rather any
type or crosslinker known in the art that is suitable to crosslink
one or more components of the first layer to one or more components
of the second layer may be used. In one embodiment, the aminated
heparin of the second layer is crosslinked to the carboxyl groups
of the HA of the first layer. In another embodiment, the aminated
heparin is crosslinked to thiol groups of the HA of the first
layer. For example, in certain embodiments, the aminated heparin is
further modified to contain NHS and a sulfhydryl-reactive malemide
group. This modified heparin can then spontaneously react with the
remaining thiol groups of the thiol-modified HA of the first layer
(FIG. 2).
[0114] In one embodiment, the heparin of the second layer extends
luminally, thereby exposing the active pentasaccharide sequence of
heparin to the blood stream when the composition is implanted. This
conformation thereby prevents immediate activation of
coagulation.
[0115] The coating of the anti-thrombogenic compositions of the
invention is biocompatible and non-toxic. For example, it is
demonstrated elsewhere herein that cells contacted to the coating
can survive and proliferate. Thus, while in certain embodiments,
the compositions of the invention are not recellularized prior to
implantation in a subject, the compositions are conducive to in
vivo recellularization of native cells. In certain embodiments, the
in vivo recellularization degrades the coating over time.
[0116] In certain embodiment, the coating of the anti-thrombogenic
compositions of the invention is non-immunogenic. That is, the
coating does not induce an immune response in the subject.
Methods of Preparing
[0117] The present invention provides a method of making
compositions having at least one surface coated with a
non-thrombogenic coating. As discussed elsewhere herein, the
composition of the invention comprises a substrate, for example a
biomaterial, tissue engineering substrate, or the like, wherein at
least one surface of the substrate is coated with a
non-thrombogenic coating. In certain embodiments, the substrate
comprises decellularized tissue. As discussed elsewhere herein, the
present invention is not limited to any particular decellularized
tissue, nor is it limited to any particular method of generating
decellularized tissue. Exemplary methods of producing
decellularized tissue are discussed elsewhere herein and are well
known in the art, see for example US2012/0064050 and WO2007/025233,
each of which are herein incorporated by reference in their
entireties.
[0118] The method comprises coating a surface of the substrate with
the non- thrombogenic coating. As discussed elsewhere herein, the
coating, in certain embodiments comprises a single layer or a
multi-layer coating. In one embodiment, the method comprises
perfusing the substrate with one or more solutions. In certain
embodiments, the decellularized tissue substrate is perfused with
water, saline, or the like, prior to application of the
non-thromobogenic coating.
[0119] As discussed elsewhere herein, in certain embodiments, the
composition of the invention comprises a hydrogel layer crosslinked
to a decellularized tissue substrate. In one embodiment, the
decellularized tissue substrate is perfused with a crosslinking
containing solution. The present invention is not limited to any
particular type of crosslinker. Rather, any suitable crosslinker
known in the art may be employed. In one embodiment, the
crosslinker is SM(PEG)n NHS-PEG-Malemide crosslinker (Thermo). In
one embodiment, the crosslinker is dissolved in DMSO and PBS to
form a crosslinking solution. The relative amount of the
crosslinker in the crosslinking solution may be varied as
appropriate. In certain embodiments, the concentration of the
crosslinker in the crosslinking solution is about 0.1 mM to about
500 mM. In another embodiment, the concentration of the crosslinker
in the crosslinking solution is about 1 mM to about 100 mM. In
another embodiment, the concentration of the crosslinker in the
crosslinking solution is about 40 mM. The crosslinker solution may
be then perfused onto the decellularized tissue substrate. As
discussed elsewhere herein, in certain embodiments the substrate is
a decellularized blood vessel. In one embodiment, the tubular
decellularized vessel is continuously perfused with the solution
through the lumen of the vessel. In one embodiment, the solution is
perfused in a closed loop fashion. In one embodiment, the substrate
is perfused with the crosslinking solution for about 5 seconds to
about 2 hours. In another embodiment, the substrate is perfused
with the crosslinking solution for about 30 seconds to about 24
hours. In another embodiment, the substrate is perfused with the
crosslinking solution for about 30 minutes.
[0120] In certain embodiments, after application of the
crosslinking solution, the substrate is perfused with a hydrogel
solution. As discussed elsewhere herein, the hydrogel solution may
comprise any suitable biopolymer, synthetic polymer, or combination
thereof. In one embodiment, the hydrogel solution comprises HA. In
one embodiment, the hydrogel solution comprises thiol-modified HA.
The hydrogel solution may be produced by dissolving the
thiol-modified HA into water or other suitable solvent. In certain
embodiments, the solvent is degassed, as in certain instances, the
HA will crosslink in the presence of oxygen. In one embodiment, the
tubular decellularized vessel is continuously perfused with the
hydrogel solution through the lumen of the vessel. In one
embodiment, the solution is perfused in a closed loop fashion. In
one embodiment, the substrate is perfused with the hydrogel
solution for about 5 seconds to about 8 hours. In another
embodiment, the substrate is perfused with the hydrogel solution
for about 30 seconds to about 4 hours. In another embodiment, the
substrate is perfused with the crosslinking solution for about 2
hours. After perfusion of the hydrogel solution, in certain
embodiments, the substrate is rinsed with water, saline, or the
like. In certain embodiments, in order to produce a rough
morphology of the luminal surface, the substrate is perfused with a
solution comprising hylaronidase and collagenase.
[0121] In one embodiment, the substrate is coated with a second
layer comprising an anti-coagulant. For example, in one embodiment,
the second layer comprises heparin or derivatives thereof. However,
the present invention is not limited to the use of heparin as an
anti-coagulant. Rather, any known anti-coagulant may be used.
Exemplary anti-coagulants include, but are not limited to vitamin K
antagonists, coumarins, curcumin (diferuloyl methane), hirudin,
heparins, Factor Xa inhibitors, direct Xa inhibitors, direct
thrombin inhibitors, natural polysaccharides and synthetic ones
based on .beta.-(1.fwdarw.4)-linked anhydroglucose units and the
like. As discussed elsewhere herein, in certain embodiments, the
heparin is modified. In one embodiment, ADH-amino modified heparin
is prepared by dissolving heparin into a suitable solvent, for
example, formamide, and adding adipic acid dihyrazide (ADH). In one
embodiment, aqueous sodium cyanoborohydride is added to the
mixture. In some embodiments, the mixture is then dialyzed. The
retentate may then be lyophilized and purified, for example, by
ethanol precipitation.
[0122] In certain embodiments, coating of the substrate with the
second layer comprises first perfusing the substrate with a second
crosslinking solution. For example, in one embodiment, the method
comprises perfusing a second crosslinking solution comprising EDC
and NHS. In one embodiment, the second crosslinking solution
comprises water, saline, or other suitable buffer. For example, in
certain embodiments the second crosslinking solution comprises
NaCl/MES buffer. In certain embodiments, the EDC/NHS of the second
crosslinking solution allows for crosslinking of the second layer
to the carboxyl groups of the HA of the first layer. In one
embodiment, the substrate is perfused with the second crosslinking
solution for about 5 seconds to about 2 hours. In another
embodiment, the substrate is perfused with the second crosslinking
solution for about 30 seconds to about 1 hour. In another
embodiment, the substrate is perfused with the second crosslinking
solution for about 15 minutes.
[0123] In certain embodiments, coating of the substrate with the
second layer comprises first activating with a crosslinking
solution the heparin (or any known anti-coagulant) before perfusion
on the substrate. For example in one embodiment the method
comprises the addition of hetero-bifunctional crosslinkers such as
Sulfo-SMCC activating the aminated heparin. This allows the
pre-activated amine groups of heparin to crosslink spontaniouslly
on accessible thiol groups on the first layer. In a second
embodiment the heparin (or any known anti-coagulant) carboxyl
groups are activated via a crosslinking solution comprising EDC and
NHS. The substrate is perfused with the second crosslinking
solution for about 5 seconds to about 2 hours. In another
embodiment, the substrate is perfused with the second crosslinking
solution for about 30 seconds to about 1 hour. In another
embodiment, the substrate is perfused with the second crosslinking
solution for about 15 minutes.
[0124] In certain embodiments, coating of the substrate with the
second layer comprises perfusing the substrate with an
anti-coagulant solution. For example, in certain embodiments, the
anti-coagulant solution comprises a heparin solution. The heparin
solution comprises heparin dissolved in a suitable solvent,
including, but not limited to, water, saline, or other buffer. For
example, in one embodiment, heparin is dissolved in NaCl/MES
buffer. As described elsewhere herein, in certain embodiments, the
heparin of the heparin solution is modified. The amount of heparin
in the heparin solution may be varied as necessary. For example,
the amount of heparin may, in certain instances, depend on the
ultimate use of the composition. In certain embodiments, the
concentration of heparin in the heparin solution is [5 .mu.M]. In
one embodiment, the substrate is perfused with the heparin solution
for about 5 seconds to about 3 hours. In another embodiment, the
substrate is perfused with the heparin solution for about 30
seconds to about 2 hours. In another embodiment, the substrate is
perfused with the second crosslinking solution for about 1 hour. In
certain embodiments, the substrate is rinsed with water, saline, or
other suitable buffer following perfusion of the heparin
solution.
[0125] Although an advantage of the present invention is that
recellularization is not required, a skilled artisan armed with the
specification would recognize that the decellularized tissue can be
recellularized if desired. Accordingly, in certain embodiments, the
method comprises ex vivo or in vitro culturing of cells on the
surface of the substrate, or on the coating of the substrate. The
cultured cells can be induced to proliferate throughout at least a
portion of the composition. For example, in certain embodiments,
cells are cultured such that they produce a confluent layer of
cells on the luminal surface of a vascular graft composition
described herein. The cells can also differentiate in vitro by
culturing the cells in differentiation. Alternatively, the cells
can differentiate in vivo when they establish contact with a tissue
within the mammal or when the cells are sufficiently close to a
tissue to be influenced by substances (e.g., growth factors,
enzymes, or hormones) released from the tissue.
[0126] As described elsewhere herein, in certain embodiments, the
substrate of the composition is decellularized tissue. Therefore,
in certain embodiments, the method comprises recellularization of
the substrate.
[0127] The number of cells that is introduced into and onto a
decellularized organ in order to generate an organ or tissue is
dependent on both the organ (e.g., which organ, the size and weight
of the organ) or tissue and the type and developmental stage of the
regenerative cells. Different types of cells may have different
tendencies as to the population density those cells will reach.
Similarly, different organ or tissues may be cellularized at
different densities. By way of example, a decellularized organ or
tissue can be seeded with at least about 1,000 (e.g., at least
10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000)
regenerative cells; or can have from about 1,000 cells/mg tissue
(wet weight, i.e., prior to decellularization) to about 10,000,000
cells/mg tissue (wet weight) attached thereto.
[0128] Cells can be introduced to a decellularized organ or tissue
by injection into one or more locations. In addition, more than one
type of cell (i.e., a cocktail of cells) can be introduced into a
decellularized organ or tissue. For example, a cocktail of cells
can be injected at multiple positions in a decellularized organ or
tissue or different cell types can be injected into different
portions of a decellularized organ or tissue. Alternatively, or in
addition to injection, regenerative cells or a cocktail of cells
can be introduced by perfusion into a cannulated decellularized
organ or tissue. For example, cells can be perfused into a
decellularized organ using a perfusion medium, which can then be
changed to an expansion and/or differentiation medium to induce
growth and/or differentiation of the regenerative cells. In the
case of a lung tissue, the cells can be introducted into either or
both of the airway compartment via the trachea, or the vascular
compartment via the pulmonary artery or vein.
[0129] During recellularization, an organ or tissue is maintained
under conditions in which at least some of the regenerative cells
can multiply and/or differentiate within and on the decellularized
organ or tissue. Those conditions include, without limitation, the
appropriate temperature and/or pressure, electrical and/or
mechanical activity, force, the appropriate amounts of O.sub.2
and/or CO.sub.2, an appropriate amount of humidity, and sterile or
near-sterile conditions. During recellularization, the
decellularized organ or tissue and the cells attached thereto are
maintained in a suitable environment. For example, the cells may
require a nutritional supplement (e.g., nutrients and/or a carbon
source such as glucose), exogenous hormones or growth factors,
and/or a particular pH.
[0130] Cells can be allogeneic to a decellularized organ or tissue
(e.g., a human decellularized organ or tissue seeded with human
cells), or regenerative cells can be xenogeneic to a decellularized
organ or tissue (e.g., a pig decellularized organ or tissue seeded
with human cells).
[0131] In some instances, an organ or tissue generated by the
methods described herein is to be transplanted into a patient. In
those cases, the cells used to recellularize a decellularized organ
or tissue can be obtained from the patient such that the
regenerative cells are autologous to the patient. Cells from a
patient can be obtained from, for example, blood, bone marrow,
tissues, or organs at different stages of life (e.g., prenatally,
neonatally or perinatally, during adolescence, or as an adult)
using methods known in the art. Alternatively, cells used to
recellularize a decellularized organ or tissue can be syngeneic
(i.e., from an identical twin) to the patient, cells can be human
lymphocyte antigen (HLA)-matched cells from, for example, a
relative of the patient or an HLA-matched individual unrelated to
the patient, or cells can be allogeneic to the patient from, for
example, a non-HLA-matched donor.
[0132] Irrespective of the source of the cells (e.g., autologous or
not), the decellularized solid organ can be autologous, allogeneic
or xenogeneic to a patient.
[0133] In certain instances, a decellularized tissue may be
recellularized with cells in vivo (e.g., after the tissue has been
transplanted into an individual). In vivo recellularization may be
performed as described above (e.g., injection and/or perfusion)
with, for example, any of the cells described herein. Alternatively
or additionally, in vivo seeding of a decellularized organ or
tissue with endogenous cells may occur naturally or be mediated by
factors delivered to the recellularized tissue.
Methods of Use
[0134] The present invention provides therapeutic methods
comprising the administration or implantation of the
anti-thrombogenic compositions (e.g., anti-thrombogenic vascular
grafts) described herein. For example, in certain embodiments, the
anti-thrombogenic vascular grafts of the invention are used in
methods to replace or bypass damaged or diseased blood vessels in a
subject. In certain embodiments, the methods are used to treat an
aneurysm in a subject. In another embodiment, the methods are used
to replace or bypass vessels which provide inadequate blood
flow.
[0135] In certain embodiments, the method comprises treating a
subject having a diseased blood vessel. For example, exemplary
diseases or disorders treated by way of the present method include,
but are not limited to, peripheral vascular disease,
atherosclerosis, aneurysm, or venous thrombosis. In one embodiment,
the subject is a mammal. In one embodiment, the subject is a
human.
[0136] Grafting of the substrates and compositions of the invention
to an organ or tissue to be augmented can be performed according to
the methods described in herein or according to art-recognized
methods. The composition can be grafted to an organ or tissue of
the subject by suturing the graft material to the target organ.
Implanting a neo-organ construct for total organ replacement can be
performed according to the methods described herein or according to
art-recognized surgical methods.
[0137] In certain embodiments vascular grafts of the invention are
sutured to existing blood vessels of the subject. For example, an
anti-thrombogenic vascular graft described herein may be sized and
shaped appropriately to mimic or replace a particular damaged or
abnormal blood vessel of the subject. In certain embodiments, the
region of native blood vessel to be replaced is surgically excised
from the subject. The ends of the vascular graft of the invention
can then be sutured to the remaining vessel.
[0138] The vascular grafts of this invention may be used in place
of any current by-pass or shunting graft, either natural or
artificial, in any application. Thus, they may be used for, without
limitation, arterial by-pass, both of the cardiac variety and that
used to treat peripheral vascular disease (PVD). A graft of this
invention may also be used as a replacement or substitute for a
fistula created for use in hemodialysis. Also the vascular graft of
the present invention can be used to replace a damaged blood vessel
such as traumatically damaged limb arteries.
[0139] In certain embodiments, the method of the invention
comprises implantation of the graft of the invention to provide an
artificial arteriovenous shunt or graft for use by dialysis
patients. In hemodyalysis, a patient's blood is "cleansed" by
passing it through a dialyzer, which consists of two chambers
separated by a thin membrane. Blood passes through the chamber on
one side of the membrane and dialysis fluid circulates on the
other. Waste materials in the blood pass through the membrane into
the dialysis fluid, which is discarded, and the "clean" blood is
re-circulated into the blood stream. Access to the bloodstream can
be external or internal. External access involves two catheters,
one placed in an artery and one in a vein. More frequently, and
preferably, internal access is provided. This is accomplished
either by an artriovenous fistula or an AV graft. An AV fistula
involves the surgical joining of an artery and a vein under the
skin. The increased blood volume stretches the elastic vein to
allow for a larger volume of blood flow. Needles are placed in the
fistula so that blood can be withdrawn for dialysis and then the
blood is returned through the dilated vein.
[0140] An AV graft may be used for people whose veins, for one
reason or another, are unsuitable for an AV fistula. An AV graft
involves surgically grafting a donor vein from the patient's own
saphenous vein, a carotid artery from a cow or a synthetic graft
from an artery to a vein of the patient. One of the major
complications with a synthetic AV graft is thrombosis and
neointimal cell proliferation that cause closure of the graft.
[0141] As described elsewhere herein, a benefit of the vascular
grafts of the invention are that they are non-thrombogenic without
the need seeding of the graft with the subject or donor cells. As
such, the grafts can be implanted at the time that it becomes
necessary. That is, there is no waiting time needed in order to
prepare the grafts. The grafts of the invention are stable during
standard refrigeration, and thus can serve as an off the shelf
composition.
EXPERIMENTAL EXAMPLES
[0142] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0143] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the present
invention and practice the claimed methods. The following working
examples therefore, specifically point out the preferred
embodiments of the present invention, and are not to be construed
as limiting in any way the remainder of the disclosure.
Example 1
Hyaluronic Acid--Heparin Based Coatings for Biological
Substrates
[0144] Described herein is the development of a coating for
decellularized biological structures (native and tissue engineered)
built from a first layer of thiol-modified hyaluronic acid (HA;
also known as hyaluronan) and a second layer of modified
heparin.
[0145] HA Coating
[0146] Thiol-modified HA (Glycosan, San Francisco, USA) was
crosslinked onto decellularized biological structures amines groups
(NH.sub.2) using the sulfhydryl (SH) groups on the HA. This was
accomplished via heterobifunctional crosslinker made up of
N-hydroxysuccinimide ester (NHS) and maleimide where NHS reacts
with the amine groups on the decellularized vessels and maleimide
reacted with the sulfhydryl groups on the hyaluronic acid. The
crosslinked hyaluronic acid created a few microns thick continuous
layer over the length of the tubular vessel, "hiding" the exposed
collagen of decellularized vessels.
[0147] Heparin Modification and Modified Heparin Coating
[0148] The second step of the coating was the "end on" aminated
heparin, produced via reductive amination, which was crosslinked
onto the carboxyl (COOH) groups of the hyaluronic acid via EDC/NHS.
The "end-on" heparin amination was accomplished on the heparin
end-chain electrophilic carbon atom, which under heat attacked the
nucleophilic nitrogen of adipic acid dihydrazide (ADH) primary
amine to yield a weak bond stabilized using sodium cyanoborohydride
(NaCNBH.sub.3). This yielded an end-on primary amine group on the
heparin (FIG. 2A Top). The end-on aminated heparin was cleaned via
dialysis, and crosslinked onto the remaining carboxyl (COOH) groups
of the HA coating via EDC/NHS. Heparin attached in this manner
extended luminally (due to hydration) exposing the active
pentasaccharide sequence of heparin preventing immediate activation
of coagulation. This reaction was schematically described in FIG.
1.
[0149] In certain instances, heparin modification comprises an
additional step. The heparin modification can be taken further by
modifying the ADH primary amine with
Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate
(Sulfo-SMCC) (FIG. 2A Bottom). This route used the ADH-amine to
link NHS leaving a sulfhydryl-reactive maleimide group. The
modified heparin was spontaneously reactive with the remaining
thiol groups on the hyaluronic coatings.
[0150] Rat Aorta Isolation
[0151] Sprague Dawley rats ascending aorta was harvested under
general anesthesia (isoflorane). Briefly, the rats were opened by a
midline laparotomy and the ascending aorta was dissected free. The
aorta was then rinsed with cold PBS and was subjected to
decellularization within half an hour of isolation.
[0152] Tissue Engineering of Vessels
[0153] Tissue-engineered porcine arteries were created by seeding
five million porcine carotid smooth muscle cells onto a tubular
polyglycolic acid mesh (3 mm in diameter and 8 mm in length;
Concordia Medical, Coventry, R.I.) around a silicone tube and
cultured in a bioreactor connected to a peristaltic pump at 5%
CO.sub.2 and 37.degree. C. The engineered vessels were harvested
from bioreactors after 8 weeks of culture and rinsed two to three
times with PBS to remove traces of culture medium. Within half an
hour of isolation, tissues were subjected to decellularization.
[0154] Decellularization Procedure of Both Rat Aortas and Tissue
Engineered Vessels
[0155] Decellularization was accomplished using a detergent-based
method that included incubation in CHAPS/SDS buffer (8 mM CHAPS, 1
M NaCl, and 25 mM ethylenediaminetetraacetic acid (EDTA), 1.8 mM
SDS, 1 M NaCl, in PBS) for 24 hours, followed by a 2-day wash with
PBS to completely remove the detergent. Finally, aortas and/or
tissue engineered grafts were incubated in PBS containing 10% (v/v)
FBS (Hyclone, Logan, Utah) and 1% Penicillin/ Streptomycin
(Pen/Strep). All decellularization steps were carried out at
37.degree. C. with agitation under sterile conditions.
Decellularized vessels were stored in PBS containing 1% Pen/Strep
at 4.degree. C. for up to 2 weeks.
[0156] Coating Protocol
[0157] The decellularized vessels were mounted on in-house built
closed loop perfusion chamber via end-ligation of the vessels onto
capped needles. Before coating the vessels, they were perfused with
5% Pen/Strep in PBS solution. The SM(PEG)n NHS-PEG-Maleimide
Crosslinker (Thermo) (100 mg) equilibrated to room temperature, was
dissolved in 187 .mu.l DMSO by vortexing followed by 2 min
sonication step. When the crosslinker-DMSO solution was clear, 3 ml
of PBS were added and total solution was immediately perfused into
the vessel via the 3-way stopcock. The crosslinker solution was
perfused back inside the vessel via a second loop, creating a
continuous perfusion in a loop fashion for 30 min. The excess
crosslinker was rinsed out of the vessel by open-end perfusion of
the vessels with 100 ml of PBS. Rinsing was not done for more than
15 min as the thiol-reactive groups of the attached crosslinker
reacted with the water in PBS. The thiol-modified hyaluronic acid
(Glycosan, San Francisco, USA) was then dissolved in 1 ml of
degased water without uncapping the vial. The vial was placed on
rotating plate for 30 minutes to fully dissolve. Two milliliters of
the reconstituted thiol-modified hyaluronic acid were perfused into
the vessel over the course of 2 hours in a closed loop fashion.
After 2 hours, the excess thiol-modified hyaluronic acid was
removed by syringe aspiration inserted into the 3-way stopcock. The
vessel was then rinsed for 2 hours with 500 ml of PBS in a one loop
fashion removing unreacted but bound hyaluronic acid.
[0158] If a rougher morphology was desired, cleaned vessels were
perfused with 5 ml of Hylaronidase (300 .mu.g/ml)/collagenase (0.5
mg/ml) mixture at 37.degree. C. for 2 hr. After perfusion of the
Hylaronidase/collagenase mixture, the vessels were rinsed with 200
ml of PBS via open-end perfusion.
[0159] ADH-amino modified heparin was prepared by adding heparin
(100 mg, 8.3 .mu.mol) into 10 mL of formamide and heating at
50.degree. C. After heparin was totally dissolved (about 30 mins),
adipic acid dihydrazide (ADH) (10 mg, 92 .mu.mol) was added. The
reaction was maintained at 50.degree. C. for 6 h. Aqueous sodium
cyanoborohydride (9.5 mg, 150 .mu.mol) was then added and the
mixture was incubated at 65.degree. C. for an additional 24 h. The
reaction mixture was diluted with 50 mL of water and dialyzed
against 2 L of water for 48 h using a 3500 molecular weight cutoff
(MWCO) dialysis membrane. The retentate was recovered, lyophilized,
and purified by ethanol precipitation. Amino-modified heparin (120
mg) was dissolved in 16% w/v NaCl/MES buffer at pH 7. EDC (110 mg)
and NHS (78 mg) was dissolved in 10 ml 16% w/v NaCl/MES buffer at
pH 5 and perfused through the vessel for 15 mins. (pre-activating
carboxyls of collagen). The excess EDC/NHS was rinsed by 50 ml
perfusion of MES buffer at pH 7. The dissolved heparin was then
perfused through the vessels for 1 hr. The excess unreacted heparin
was washed by perfusion with 500 ml of PBS.
[0160] Characteristics of HA and HA-Heparin Coatings
[0161] The crosslinking of HA onto decellularized aortas using the
aortas amine groups and HA thiol groups via NHS-maleimide
SM(PEG).sub.12 crosslinker was optimized to 40 mM SM(PEG).sub.12
crosslinker concentration for a full coverage of the decellularized
structures. The surface accessible carboxyl groups available on the
HA layer for the heparin addition step was assessed via
nanoparticles (NPs) tagging. As indicated by the NPs at 40 mM
SM(PEG).sub.12 concentration, an abundance of free carboxyl groups
remain on the surface of the HA coating.
[0162] Tubular decellularized rat abdominal aortas were coated in a
closed loop perfusion. The morphological changes in the vessel
luminal side were obvious in scanning electron microscopy (SEM)
images, as shown in FIG. 4. The control-decellularized aortas
(luminal diameter about 3 mm) have a rough appearance due to the
decellularization detergent washes necessary for cellular removal.
HA crosslinking via SM(PEG).sub.12 onto the vessels surface
resulted in a smooth vessel surface. The "end-on" crosslinked
heparin layer on top of the HA layer restored the rougher
appearance of the vessel.
[0163] A SEM cross-section of the decellularized rat abdominal
aortas layer-by-layer HA-heparin coating showed that the coating
extending luminally in the aorta. As shown in FIG. 5, the coating
was observed as a smoothed structure coating the porous vessel.
[0164] Histological analyses of layer-by-layer coated vessels
cross-sections were performed with Toluidine Blue, Alician Blue,
and Alican Blue/PAS. Toluidine Blue is a basic dye attracted to
negatively charged structures such as heparin. Alcian Blue and
Alcian blue/PAS cationic dyes are also attracted to negatively
charged structures under alkaline conditions. As shown in FIG. 6,
all three dyes stained the HA-heparin coating strongly.
[0165] To assess the thrombogenic effects of the coatings, isolated
blood platelets were incubated under constant agitation on the
surface of decellularized, HA-coated and HA-heparin coated vessels.
As shown by the SEM images in FIGS. 7 and 8, platelets strongly
adhered to uncoated decellularized vessels and the surface of the
uncoated decellularized vessels were densely covered in platelets
and red blood cells. Decellularized vessels contain abundant
collagen on the surface, which is a potent activator of platelets,
and thus uncoated decellularized vessels are thrombogenic. In
comparison, both HA and HA-heparin coated vessels strongly resisted
platelet adhesion. In fact, it was hard to find any platelets on
the surface of the coated vessels.
[0166] To assess the amount of immobilized functional heparin on
the HA layer, the Factor X assay was utilized. It was a method
based on the conformational change of antithrombin III by bioactive
heparin, resulting in factor Xa inhibition. The Factor X inhibition
was then measured by S-2732 Chromogenic substrate
(Suc-Ile-Glu(g-Pip)-Gly-ArgpNA), and functional heparin was
assessed by comparison with Heparin standards (0-100 ng) reacted in
the same manner as the scaffolds. The capacity of a surface to
inactivate Factor X was strongly correlated with the surface
capacity to delay the blood coagulation cascade. In comparison with
freshly excised native aorta lined by functional endothelial cells
to retard coagulation, the HA-heparin coated decellularized aortas
showed at least as much active heparin, which inhibits Factor X
activity (FIG. 9). Decellularized control aortas were highly
thrombogenic and did not demonstrate any heparin activity, as
demonstrated by very low Factor X inactivation (see FIG. 9). A
continuous monolayer of human umbilical vein endothelial cells
(HUVEC) grown for 5 days which was known to demonstrate
anticoagulant activity demonstrated lower anti Factor X activity
than the HA-heparin coatings.
[0167] To assess the potential toxicity of these coatings, human
umbilical vein endothelial cells (HUVECs) isolated from human
umbilical cord were seeded onto the HA-heparin layer-by-layer
coated aortas and cultured for 2 weeks. The HUVECs proliferated
over the coating surface but also invaded the coating by degrading
it over the course of the two weeks. The HUVECs downward invasion
into the coating could be seen from the confocal microscopy image
(FIG. 10), where inward migrating HUVECs were shown over a 10 .mu.m
z-stack.
[0168] Experiments were also performed to examine whether the
coatings produce inflammatory responses, stimulate recruitment of
monocytes and macrophages, or stimulate adhesion and invasion of
leukocytes. Further, experiments were done to determine whether
grafts coated with these coatings became luminally coated with host
endothelial cells after implantation into the vascular system.
Further, experiments were conducted to determine if the HA layer or
heparin layer bound to growth factors that were conducive to the
cellular repopulation of the grafts. Finally, experiments were
performed to determine if the coatings were resistant to intimal
hyperplasia, a common mid-term to late-term failure mode for
arterial grafts.
[0169] The data described herein demonstrated HA and HA-heparin
coatings served as anti-thrombogenic coatings for biological
scaffolds including decellularized vascular grafts. The coating
described herein utilized the crosslinking of HA to protein
substrates of the decellularized tissue. Further, the
layer-by-layer coating of heparin on HA enhanced the immediate
anti-coagulant properties. The HA layer offered a physical barrier
to thrombogenic collagen, other extracellular matrix proteins, or
synthetic materials which stimulated the extrinsic or intrinsic
coagulation cascade, while the layer of active heparin was attached
in its active conformation with an exposed pentasaccharide sequence
free to interact with blood components.
[0170] It was demonstrated that coating with HA alone inhibited
coagulation as evidenced by attenuation of platelet
adhesion/activation and inactivation of Factor X and thrombin
activity. Thus, HA coatings may be efficacious for small caliber
grafts (i.e., less than or equal to 6 mm in diameter). Further,
HA-coated grafts displayed higher mechanical properties as measured
by increased suture strengths that were conferred by the mechanical
characteristics of the coating. The data also demonstrated that
HA-coatings and HA-heparin layer-by-layer coatings were highly
conducive to cellular ingrowth. Therefore, as presented herein, HA
coatings and layer-by-layer HA-heparin coatings served as
functional anti-thrombotic coatings of vascular grafts.
[0171] In order to more fully characterize the process of gel
formation on the surface of collagen-containing grafts, the time of
gelation of the PEG crosslinker and hyaluronic acid (HA) was
evaluated at 25.degree. C. via a strain-controlled rheometer (ARES
LS1, TA Instruments, New Castle, Del.). A porcine decellularized
aorta was mounted onto the titanium cone of the apparatus, followed
by the incubation of 400 .mu.l of PEG crosslinker for 45 mins.
After this, the PEG crosslinker was aspirated, and 1000 .mu.l of HA
was loaded on top of the decellularized porcine aorta (these steps
mimicked the 3D perfused coating). Finally, the decellularized
porcine aorta-HA gel complex was closed with the stainless plate of
the apparatus (25-mm diameter, 0.04-radian angle, 45-.mu.m gap). As
a control, the HA gels were deposited onto decellularized porcine
aorta without the addition of the PEG crosslinker step, and the
system was closed with the stainless plate in the same way. Elastic
(G') and loss (G'') moduli (1% strain, 1 Hz) were recorded every 9
s for 24 h. The complex shear modulus (storage modulus) G of the
HA-PEG gels and HA gels alone was calculated from:
G = Td 2 I p r ##EQU00001##
where T was the torque response, d was the sample diameter, y was
the sinusoidal shear strain, and I.sub.p was the polar moment of
inertia of the cylinder (I.sub.p=.pi.d.sup.4/32).
[0172] As can be seen in FIG. 11, the rheological response to
imposed oscillatory shear stress of the HA gels on the
decellularized aorta in the absence of the crosslinker was a
fluid-like initial response that polymerized to 80% of the added
volume after 23 hours. This was shown in panel A and the 23 hours
were indicated by the red dotted line intersecting the x-axis of
log time at 5.times.10.sup.4 sec. When transforming the time frame
from a Log scale into a Ln scale (Ln scale plotted in panel B), the
polymerization of HA component alone on the decellularized graft
resulted in the same time frame of 23 hours to gain 80%
polymerization.
[0173] The addition of the PEG crosslinker on the decellularized
aorta activating the amine groups before the addition of the HA
component induced 80% polymerization of the HA layer within 4 hours
of incubation. This was evident in both panels C and D where in
both Log time and Ln time plots (respectively) of the storage
modulus (G'') with respect to time becomes constant at panel C
10.sup.4 sec (log scale).
[0174] The addition of the PEG crosslinker on the decellularized
aorta activating the amine groups before the addition of the HA
component induced 80% polymerization of the HA layer within 4 hours
of incubation. The absence of large magnitude changes in the
viscous (G'') and elastic (G') properties after 4 hours is
indicative of no further microstructural change in the HA gel
component indicating that HA preceded by PEG attains its `mature`
form within 4 hours. The 80% polymerization in all cases was
estimated roughly as the place where no further microstructural
changes were evident from the constancy in storage modulus with
respect to time. This was identified in all panels with a red
dotted line.
[0175] The rheological characterization thereby described set the
perfusion time of the HA component onto the decellularized vessels
to create the coating to a minimum perfusion time of 4 hours to a
maximum perfusion time of 18 hours.
[0176] To asses the stability of the coatings, porcine and rat
aorta, as well as tissue engineered vascular grafts, were
decellularized and coated both with Hyaluronic acid (HA) alone and
Hyaluronic acid/Heparin (HA/HP). The example shown here was from
rat decellularized abdominal aortas but the various studied
vasculature structures behaved similarly.
[0177] The stability of HA and HA/HP coatings was evaluated at
37.degree. C. over two weeks by incubating the coated
decellularized vessels under the following conditions: 1) PBS; 2)
M199 cell culture medium; and 3) freshly isolated rat blood plasma
obtained by filtering freshly drawn rat blood through 0.2 .mu.m
syringe filters. Following the above described incubations, the
eluted coating was assessed by quantifying the total amount of
polysaccharides in solution at Day 1, 3, 7 and 14 using the
Carbazole assay. The Carbazole assay quantifies polysaccharides in
solution based on colorimetric changes with a detection limit of 2
.mu.g/ml.
[0178] The amount of released HA and HA/HP from the various coated
decellularized surfaces was comparable to the amount of
polysaccharides released from the uncoated decellularized vessels
(stable negative control samples). For instance the highest amounts
of detected released polysaccharides from the coated surfaces was
of 0.2 .mu.g/ml which was the amount of polysaccharides released by
the non-coated decellularized vessel used as negative control and
it also was below the Carbazole assay detection limit. The low
polysaccharides released into solution over time indicated high
stability of the coating under in vitro physiological
conditions.
[0179] Following the two weeks incubation time, the coated
decellularized grafts were histologically assessed for remaining
coating on the decellularized vessels using Toluidine Blue, and
Alican Blue/PAS dyes. As shown in FIG. 12, following PBS incubation
at 37.degree. C. for two weeks the Hyaluronic acid/Heparin coated
decellularized rat aortas kept a continuous layer of the coating in
place. This was especially evident in comparison with the uncoated
(control) decellularized rat aortas where only the background stain
of the decellularized aorta was present. In addition, after two
weeks incubation with freshly drawn rat blood plasma (changed every
three days) the Hyaluronic acid/ Heparin coating layer also
remained visible on the decellularized rat aortas as seen by
Toluidine Blue, and Alican Blue/PAS stains. This observation
suggested that blood enzymes did not degrade the coating in its
entity in two weeks time.
[0180] The endothelial cell growth response on the individual
coating layers was evaluated. A PEG crosslinker layer alone, PEG
crosslinker and Hyaluronic acid layer, and lastly the PEG
crosslinker-Hyaluronic acid and heparin components combined were
coated. After gelation, each of these systems was seeded with
freshly isolated human umbilical vein endothelial cells (HUVECs)
and cultured for three days. FIG. 13 shows the day 3 cultured
HUVECs that were stained with DAPI for nuclei (FIG. 13), and
VE-Cadherin for an endothelial membrane surface marker (FIG. 13).
It was seen that the various layers of the coating supported
endothelial cell adhesion and proliferation in vitro. Certain areas
of the coating had less dense cell coverage than other areas, as
the cell coverage was not uniform throughout the coating.
[0181] Rat aortas from the thoracic and abdominal portion were
harvested and decellularized. The decellularized rat aortas were
implanted in three rats without further modification (control
group) and three decellularized rat aortas were Hyaluronic Acid
coated prior to implantation, using the steps of applying the PEG
crosslinker followed by thiolated HA to form a gel on the luminal
surface of the decellularized aortas. The rat implantation was done
by first clamping the proximal and distal portions of the
infrarenal aorta and removing a 17-mm segment of aorta that was
replaced by decellularized rat aorta untreated (control group) or
Hyaluronic Acid-coated decellularized rat aorta. The grafts were
inserted by end-to-end anastomosis using interrupted 9-0
monofilament nylon sutures. Following the suturing of the grafts in
place the distal and then the proximal vascular clamps were slowly
removed, and flow was restored through the graft. The graft patency
was monitored by color Doppler imaging and pulse waves recorded
with a 12-MHz sector probe and an echo-imaging apparatus at 2 and 4
weeks. Graft diameter and blood flow velocity were measured. Signs
of thrombosis and aneurysm formation were carefully checked. The
rats were sacrificed after four weeks and the grafts were evaluated
histologically by staining segments of the grafts using hematoxylin
and eosin (H&E) for general evaluation.
[0182] Uncoated decellularized grafts (control groups) shown in
FIG. 14 top panel formed large clots with large amounts of fibrin
deposition that almost fully occluded the blood flow of the
abdominal aorta. The H&E staining was showing the large
fibrinized blood clot that was covering almost the entire luminal
opening of the implanted graft. The Doppler ultrasound recording of
the blood flow at four weeks post-implantation was showing no
recording of the blood flow. In some rats there was total absence
of blood flow and in other there was some minimal blood flow of
about 3 cm/s. On the other hand, the Hyularonic acid coated
decellularized rat aorta demonstrated absence of blood clots in
most rats as was shown in the H&E stained cross section of the
bottom panel in FIG. 13. Some rats showed small to medium sized
clots deposition and some occlusion of the implanted aorta. The
Hyaluronic acid coating was visible in the explants on some areas
of the lumen (shown in FIG. 14 bottom panel) but the thickness of
the coating was greatly reduced. The Hyaluronic acid coated
decellularized rat aortas demonstrated normal blood flow at the
time of explantation of 4 weeks as was shown in FIG. 15 bottom
panel. The explants were not dilated and resembled in appearance as
pre-implantation. On most areas, the implants were abluminally
integrated within the surrounding tissues. Staining for T-cells and
macrophage markers has revealed no conclusive signs of inflammation
reactions in the implants.
[0183] Hyaluronic acid coated decellularized vascular rat aortas
implanted in the rat animal model suggested clinical feasibility of
hyaluronic acid coated vascular grafts.
[0184] Tissue engineered vascular grafts (TEVG) were grown as per
established protocols starting with dog harvested smooth muscle
cells. Grafts were 4 mm diameter and approximately 5 cm in length.
TEVG were decellularized as described previously in patent. In the
control group, the decellularized TEVG were implanted without
further modifications; and the second animal group received
Hyaluronic acid and heparin-coated decellularized TEVG. The dog
study used longer grafts than the rat animal study (implanted
grafts were about 5 cm in length), and the implantation site of
carotid artery was chosen as a more aggressive animal model than
the rat abdominal aorta.
[0185] The implantation surgery was performed by first identifying
and dissecting the dog carotid arteries free from surrounding
tissue. One centimeter of the carotid artery was removed and a
Hyaluronic acid-heparin-coated decellularized TEVG (4-mm internal
diameter) was implanted into the right carotid artery using end to
side anastomoses. The non-coated decellularized TEVG (4-mm internal
diameter) was implanted in the left carotid artery by the same
procedure. The internal diameter of the vascular grafts closely
matched with the recipient carotid arteries. The patency of all
vascular grafts was checked by Doppler ultrasound immediately after
implantation. The implants were kept in the dogs for four weeks, at
which time they were explanted and histologically examined.
[0186] Similar to the rat animal model, the non-coated
decellularized TEVG (control group) shown in FIG. 16 top panel
formed large blood clots with large amounts of fibrin deposition
and became almost fully occluded. The H&E stain in FIG. 15
showed the large fibrinized blood clot that was covering almost the
entire luminal opening of the implanted graft. Hyaluronic
acid-heparin-coated decellularized TEVG demonstrated variable
results, with some areas showing total absence of blood clots and
other areas showing some blood clot formation, but significantly
less than the control grafts. Endothelial cells were found on the
luminal surface of the Hyaluronic coated TEVG as identified in FIG.
16.
[0187] Hyaluronic acid coated decellularized TEVG implanted in the
dog carotid artery animal model was a challenging model that
further suggest clinical feasibility of hyaluronic acid coated
vascular grafts.
[0188] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While this invention has
been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention
may be devised by others skilled in the art without departing from
the true spirit and scope of the invention. The appended claims are
intended to be construed to include all such embodiments and
equivalent variations.
* * * * *