U.S. patent application number 13/480205 was filed with the patent office on 2012-11-29 for sterilized, acellular extracellular matrix compositions and methods of making thereof.
Invention is credited to ROBERT G. MATHENY.
Application Number | 20120302499 13/480205 |
Document ID | / |
Family ID | 47219630 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120302499 |
Kind Code |
A1 |
MATHENY; ROBERT G. |
November 29, 2012 |
STERILIZED, ACELLULAR EXTRACELLULAR MATRIX COMPOSITIONS AND METHODS
OF MAKING THEREOF
Abstract
Methods for sterilizing and decellularizing extracellular matrix
materials are disclosed. Extracellular matrix compositions produced
using the disclosed methods are also disclosed.
Inventors: |
MATHENY; ROBERT G.;
(Norcross, GA) |
Family ID: |
47219630 |
Appl. No.: |
13/480205 |
Filed: |
May 24, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61490693 |
May 27, 2011 |
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61490873 |
May 27, 2011 |
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61491723 |
May 31, 2011 |
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61650911 |
May 23, 2012 |
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Current U.S.
Class: |
514/8.9 ; 422/33;
514/9.1; 977/700; 977/915 |
Current CPC
Class: |
A61L 27/3633 20130101;
A61L 27/3691 20130101; A61L 2430/20 20130101; A61L 2/0094 20130101;
A61F 2/2412 20130101; A61L 27/3882 20130101; A61L 27/3687 20130101;
A61L 2430/40 20130101; A61L 27/54 20130101; A61L 27/3873 20130101;
A61F 2/2415 20130101; A61F 2/24 20130101 |
Class at
Publication: |
514/8.9 ;
514/9.1; 422/33; 977/700; 977/915 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61L 2/20 20060101 A61L002/20 |
Claims
1. A method for sterilizing and rendering acellular an
extracellular matrix (ECM) material, comprising: positioning an ECM
material within an interior space of a reactor vessel; introducing
carbon dioxide into the interior space of the reactor vessel at
supercritical pressure and temperature conditions, thereby
sterilizing the ECM material; and rapidly depressurizing the
interior space of the reactor vessel at a depressurization rate of
at least 400 psi/minute, thereby rendering the ECM material
acellular.
2. The method of claim 1, wherein the depressurization rate ranges
from about 400 psi/minute to about 2,600 psi/minute.
3. The method of claim 1, wherein the depressurization rate ranges
from about 700 psi/minute to about 1,500 psi/minute.
4. The method of claim 1, wherein the depressurization rate ranges
from about 1,000 psi/minute to about 1,200 psi/minute.
5. The method of claim 1, further comprising introducing at least
one secondary sterilant into the interior space of the reactor
vessel.
6. A sterilized, acellular extracellular matrix (ECM) composition
produced using the method of claim 1.
7. The sterilized, acellular ECM composition of claim 6, wherein
the ECM composition comprises at least one tissue, each tissue of
the at least one tissue being from a respective tissue source,
wherein the tissue source of each tissue of the at least one tissue
is selected from the group consisting of small intestinal
submucosa, stomach submucosa, large intestinal tissue, urinary
bladder submucosa, liver basement membrane, pericardium,
epicardium, endocardium, myocardium, lung tissue, kidney tissue,
pancreatic tissue, prostate tissue, mesothelial tissue, fetal
tissue, a placenta, a ureter, veins, arteries, heart valves with or
without their attached vessels, tissue surrounding the roots of
developing teeth, and tissue surrounding growing bone.
8. The sterilized, acellular ECM composition of claim 6, wherein
the ECM composition is at least about 96% decellularized.
9. A method for sterilizing, rendering acellular, and incorporating
an additive into an extracellular matrix (ECM) material,
comprising: positioning an ECM material within an interior space of
a reactor vessel; introducing carbon dioxide into the interior
space of the reactor vessel at supercritical pressure and
temperature conditions, thereby sterilizing the ECM material;
rapidly depressurizing the interior space of the reactor vessel at
a depressurization rate of at least 400 psi/minute, thereby
rendering the ECM material acellular; and introducing one or more
additives into the interior space of the reactor vessel, whereby at
least a portion of each additive of the one or more additives is
incorporated into the sterilized and acellular ECM material.
10. The method of claim 9, wherein the depressurization rate ranges
from about 400 psi/minute to about 2,600 psi/minute.
11. The method of claim 9, wherein the depressurization rate ranges
from about 700 psi/minute to about 1,500 psi/minute.
12. The method of claim 9, wherein the depressurization rate ranges
from about 1,000 psi/minute to about 1,200 psi/minute.
13. The method of claim 9, further comprising introducing at least
one secondary sterilant into the interior space of the reactor
vessel.
14. The method of claim 9, wherein the one or more additives
comprises at least one growth factor.
15. The method of claim 9, wherein the one or more additives
comprises at least one cytokine.
16. The method of claim 9, wherein the one or more additives
comprises at least one proteoglycan.
17. The method of claim 9, wherein the one or more additives
comprises at least one glycosaminoglycan (GAG).
18. The method of claim 9, wherein the one or more additives
comprises at least one of a protein, a peptide, and a nucleic
acid.
19. The method of claim 9, wherein the one or more additives
comprises at least one pharmaceutical agent.
20. The method of claim 9, wherein the one or more additives
comprises nanoparticles.
21. The method of claim 9, wherein the step of introducing the one
or more additives into the interior space of the reactor vessel is
performed contemporaneously with the step of rapidly depressurizing
the interior space of the reactor vessel.
22. A sterilized, acellular extracellular matrix (ECM) composition
produced using the method of claim 9.
23. The sterilized, acellular ECM composition of claim 22, wherein
the ECM composition comprises at least one tissue, each tissue of
the at least one tissue being from a respective tissue source,
wherein the tissue source of each tissue of the at least one tissue
is selected from the group consisting of small intestinal
submucosa, stomach submucosa, large intestinal tissue, urinary
bladder submucosa, liver basement membrane, pericardium,
epicardium, endocardium, myocardium, lung tissue, kidney tissue,
pancreatic tissue, prostate tissue, mesothelial tissue, fetal
tissue, a placenta, a ureter, veins, arteries, heart valves with or
without their attached vessels, tissue surrounding the roots of
developing teeth, and tissue surrounding growing bone.
24. The sterilized, acellular ECM composition of claim 22, wherein
the ECM composition is at least about 96% decellularized.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing dates of
U.S. Provisional Patent Application No. 61/490,693, filed on May
27, 2011, U.S. Provisional Patent Application No. 61/490,873, filed
on May 27, 2011, U.S. Provisional Patent Application No.
61/491,723, filed on May 31, 2011, and U.S. Provisional Patent
Application No. 61/650,911, filed on May 23, 2012, each of which is
hereby incorporated by reference herein in its entirety.
FIELD
[0002] The invention generally relates to sterilized, acellular
extracellular matrix compositions and methods of making such
compositions. More particularly, the invention relates to methods
of contemporaneously sterilizing and decellularizing extracellular
matrix compositions, as well as the sterilized, acellular
compositions resulting from such methods.
BACKGROUND
[0003] Conventional techniques for sterilizing tissue compositions
often alter the properties of the tissue compositions and/or damage
important components of the tissue compositions, such as growth
factors. Consequently, these conventional sterilization techniques
often render tissue compositions unfit for their intended purposes.
For example, ethylene oxide is a toxic, mutagenic, and carcinogenic
substance that can weaken tissue compositions, reduce the growth
factor content of tissue compositions, and denature proteins within
tissue compositions. Similarly, conventional steam sterilization
techniques are incompatible with the biopolymers of tissue
compositions, and gamma radiation causes significant decreases in
the integrity of tissue compositions. Although there are known
techniques for sterilizing tissue compositions without altering the
properties of the tissue compositions, many of these techniques,
such as anti-bacterial washes, often fail to completely sterilize
the tissue compositions and/or leave residual toxic contaminants in
the tissue compositions.
[0004] Additionally, when tissue compositions are designed for
implantation within the body of a subject, the tissue compositions
must often be exposed to a separate, time-consuming
decellularization process. This decellularization process is
intended to remove cells from the tissue compositions, thereby
decreasing the likelihood that the subject's immune system will
reject the implanted tissue compositions and/or generate a
significant inflammatory response. However, conventional
decellularization techniques merely decellularize portions of the
tissue compositions such that native cells remain in the tissue
compositions following the decelluarization process.
[0005] U.S. Pat. No. 7,108,832 (the '832 patent), which is assigned
to NovaSterilis, Inc., discloses a method that sterilizes various
materials through the use of supercritical carbon dioxide. However,
as with other known sterilization methods, tissue compositions that
are sterilized using the process disclosed in the '832 patent must
be exposed to a separate decellularization process, as described
above.
[0006] Accordingly, there is a need in the art for a single method
of sterilizing and decellularizing a tissue composition, such as an
extracellular matrix composition. More particularly, there is a
need in the art for a single method of (a) sterilizing a tissue
composition while maintaining the native properties of the tissue
composition and (b) decellularizing the tissue composition such
that the tissue composition is acellular. There is still a further
need for a method of incorporating additives into a tissue
composition during sterilization and/or decellularization of the
tissue composition.
SUMMARY
[0007] Methods for sterilizing and decellularizing an extracellular
matrix (ECM) material are disclosed. In one aspect, the methods
include harvesting of a selected ECM tissue, freezing the selected
ECM tissue, thawing the selected ECM tissue, and isolating an ECM
material. The isolated ECM material is subjected to incubation and
rinsing before it is processed in supercritical carbon dioxide and
subsequently exposed to rapid depressurization. During or after the
rapid depressurization of the ECM material, one or more additives
can be incorporated into the ECM material to impart desired
characteristics to the resulting ECM composition. Sterilized,
acellular ECM compositions produced using the disclosed methods are
also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] These and other features of the preferred embodiments of the
invention will become more apparent in the detailed description in
which reference is made to the appended drawings wherein:
[0009] FIGS. 1-2 depict the results of an experiment in which DNA
content was measured for small intestinal submucosa (SIS)
compositions following various sterilization methods, including the
sterilization methods described herein. FIG. 1 shows the DNA
content of each SIS composition following sterilization. FIG. 2
shows the percentage of DNA that was removed from each SIS
composition following sterilization, as compared to raw,
unprocessed SIS.
[0010] FIGS. 3-4 depict the results of an experiment in which
native growth factor content was measured for SIS compositions
following various sterilization methods, including the
sterilization methods described herein. FIG. 3 shows the bFGF
content of each SIS composition (normalized by dry weight of
samples) following sterilization. FIG. 4 shows the active
TGF-.beta. content of each SIS composition (normalized by dry
weight of samples) following sterilization.
[0011] FIG. 5 depicts the results of an experiment in which bFGF
was incorporated into SIS compositions during rapid
depressurization, as described herein. FIG. 5 shows the bFGF
content for each SIS composition (normalized by dry weight of
samples) following rapid depressurization.
[0012] FIG. 6 depicts the results of an experiment in which the
tensile strength of two-ply SIS compositions was measured following
various sterilization methods, including the sterilization methods
described herein. FIG. 6 shows the tensile strength measured for
each SIS composition following sterilization.
[0013] FIG. 7 depicts the results of an experiment in which native
growth factor content was measured for SIS compositions following
various sterilization and/or decellularization methods, including
the sterilization and decellularization methods described herein.
FIG. 7 shows the bFGF enzyme-linked immunosorbent assay (ELISA)
results for each SIS composition (normalized by dry weight of
samples) following sterilization and/or decellularization.
[0014] FIG. 8 shows the DNA content in SIS after it is processed in
various ways. The baseline measurement is raw. The tissue was then
exposed to supercritical CO.sub.2 followed by rapid
depressurization (RDP) to facilitate enhanced removal of DNA and
cellular debris. After the RDP, the tissue was placed in
supercritical CO.sub.2 with peracetic acid (PAA) for sterilization.
The comparison is to processed SIS either unsterilized or
sterilized with ethylene oxide (ETO).
[0015] FIG. 9 shows the Percent removal of DNA from SIS after it is
processed in various ways. The baseline measurement is raw. The
tissue was then exposed to supercritical CO.sub.2 followed by rapid
depressurization (RDP) to facilitate enhanced removal of DNA and
cellular debris. After the RDP, the tissue was placed in
supercritical CO.sub.2 with peracetic acid (PAA) for sterilization.
The comparison is to processed SIS either unsterilized or
sterilized with ethylene oxide (ETO).
[0016] FIG. 10 shows the variable active Transforming Growth Factor
(TGF-beta) content in SIS after it is processed in various ways.
The baseline measurement is raw, or unprocessed SIS followed by
processing with only Triton X-100 (TX-100) detergent. The tissue
was then exposed to supercritical CO.sub.2 followed by rapid
depressurization (RDP) to facilitate enhanced removal of DNA and
cellular debris. After the RDP, the tissue was placed in
supercritical CO.sub.2 with peracetic acid (PAA) for sterilization.
The comparison is to processed SIS either unsterilized or
sterilized with ethylene oxide (ETO).
[0017] FIG. 11 shows the variable basic Fibroblast Growth Factor
(bFGF) content in SIS after it is processed in various ways. The
baseline measurement is raw, or unprocessed SIS followed by
processing with only Triton X-100 (TX-100) detergent. The tissue
was then exposed to supercritical CO.sub.2 followed by rapid
depressurization (RDP) to facilitate enhanced removal of DNA and
cellular debris. After the RDP, the tissue was placed in
supercritical CO.sub.2 with peracetic acid (PAA) for sterilization.
The comparison is to processed SIS either unsterilized or
sterilized with ethylene oxide (ETO).
[0018] FIG. 12 shows the addition of basic Fibroblast Growth Factor
(bFGF) content to SIS using rapid depressurization. The baseline
measurement is raw, or unprocessed SIS. The comparison is to
processed SIS either unsterilized or sterilized with ethylene oxide
(ETO).
DETAILED DESCRIPTION
[0019] The present invention may be understood more readily by
reference to the following detailed description, examples, and
claims, and their previous and following description. However,
before the present devices, systems, and/or methods are disclosed
and described, it is to be understood that this invention is not
limited to the specific devices, systems, and/or methods disclosed
unless otherwise specified, as such can, of course, vary. It is
also to be understood that the terminology used herein is for the
purpose of describing particular aspects only and is not intended
to be limiting.
[0020] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to a "leaflet" can include two or more such leaflets
unless the context indicates otherwise.
[0021] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0022] As used herein, the terms "optional" and "optionally" mean
that the subsequently described event or circumstance may or may
not occur, and that the description includes instances where said
event or circumstance occurs and instances where it does not.
[0023] The word "or" as used herein means any one member of a
particular list and also includes any combination of members of
that list.
[0024] Unless otherwise expressly stated, it is in no way intended
that any method or aspect set forth herein be construed as
requiring that its steps be performed in a specific order.
Accordingly, where a method claim does not specifically state in
the claims or descriptions that the steps are to be limited to a
specific order, it is in no way intended that an order be inferred,
in any respect. This holds for any possible non-express basis for
interpretation, including matters of logic with respect to
arrangement of steps or operational flow, plain meaning derived
from grammatical organization or punctuation, or the number or type
of aspects described in the specification.
[0025] Without the use of such exclusive terminology, the term
"comprising" in the claims shall allow for the inclusion of any
additional element--irrespective of whether a given number of
elements is enumerated in the claim, or the addition of a feature
could be regarded as transforming the nature of an element set
forth in the claims. Except as specifically defined herein, all
technical and scientific terms used herein are to be given as broad
a commonly understood meaning as possible while maintaining claim
validity.
[0026] As used herein, a "subject" is an individual and includes,
but is not limited to, a mammal (e.g., a human, horse, pig, rabbit,
dog, sheep, goat, non-human primate, cow, cat, guinea pig, or
rodent), a fish, a bird, a reptile or an amphibian. The term does
not denote a particular age or sex. Thus, adult and newborn
subjects, as well as fetuses, whether male or female, are intended
to be included. A "patient" is a subject afflicted with a disease
or disorder. The term "patient" includes human and veterinary
subjects. As used herein, the term "subject" can be used
interchangeably with the term "patient."
[0027] As used herein, the term "acellular" is meant to describe
extracellular matrix compositions that are at least 80%
decellularized such that the extracellular matrix composition is
80% without cells and/or cellular remnants. In some exemplary
aspects described herein, the term "acellular" can refer to
extracellular matrix compositions that are at least 90%
decellularized such that the extracellular matrix composition is at
least 90% without cells and/or cellular remnants. In other
exemplary aspects described herein, the term "acellular" can refer
to extracellular matrix compositions that are at least 95%
decellularized such that the extracellular matrix composition is at
least 95% without cells and/or cellular remnants. In other
exemplary aspects described herein, the term "acellular" can refer
to extracellular matrix compositions that are at least 96%
decellularized such that the extracellular matrix composition is at
least 96% without cells and/or cellular remnants. In still other
exemplary aspects described herein, the term "acellular" can refer
to extracellular matrix compositions that are at least 97%
decellularized such that the extracellular matrix composition is at
least 97% without cells and/or cellular remnants. In further
exemplary aspects described herein, the term "acellular" can refer
to extracellular matrix compositions that are at least 98%
decellularized such that the extracellular matrix composition is at
least 98% without cells and/or cellular remnants. In still further
exemplary aspects described herein, the term "acellular" can refer
to extracellular matrix compositions that are at least 99%
decellularized such that the extracellular matrix composition is at
least 99% without cells and/or cellular remnants. Thus, as used
herein, the term "acellular" can refer to extracellular matrix
compositions that are decellularized at levels of 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 100%, and any percentages falling between these
values.
[0028] As used herein, the term "additive" refers to materials that
can be selectively incorporated into the disclosed ECM materials to
impart predetermined properties to the sterilized, acellular ECM
compositions disclosed herein. Such additives can include, for
example and without limitation, growth factors, cytokines,
proteoglycans, glycosaminoglycans (GAGs), proteins, peptides,
nucleic acids, small molecules, cells and pharmaceutical agents,
such as statin drugs, corticosterioids, anti-arrhythmic drugs,
nonsteroidal anti-inflammatory drugs, other anti-inflammatory
compounds, nanoparticles, and metallic compounds.
[0029] As used herein, the term "contemporaneously" refers to the
simultaneous and/or overlapping occurrence of events, as well as
the sequential occurrence of events within about thirty minutes
before or after one another. Thus, if a first event occurs, then a
second event can be said to have occurred contemporaneously with
the first event if it occurred concurrently with the first event or
within thirty minutes before or after the first event. For example,
if a first method step is performed, then a second method step
performed five minutes after the first method step can be said to
be performed "contemporaneously" with the first method step.
Similarly, if the second method step was performed ten minutes
before performance of a third method step, then the second method
step can be said to be performed "contemporaneously" with the third
method step.
[0030] As used herein, the term "emulsion" refers to a mixture in
which a first ECM material is dispersed within a second ECM
material, with the first ECM material being immiscible with the
second ECM material. The "emulsions" described herein can refer to
either oil-in-water type emulsions or water-in-oil type
emulsions.
[0031] As used herein, the term "suspension" refers to mixture in
which a solid ECM material, such as, for example and without
limitation, particulate ECM, is dispersed (suspended) in a fluid
ECM material, such as, for example and without limitation, ECM gel
or ECM liquid.
[0032] As used herein, the term "supercritical" refers to a fluid
state of a material when it is held at or above its critical
temperature and critical pressure. When a material is held at or
above its critical temperature and critical pressure, then it
typically adopts functional properties of both a gas and a liquid
and is said to function as a supercritical fluid. Thus, for
example, when carbon dioxide is held at or above its critical
temperature (31.1.degree. C.) and its critical pressure (1,071
psi), it behaves as a supercritical carbon dioxide fluid and can,
for example, exhibit the expansion properties of a gas while having
the density of a liquid.
[0033] Described herein are sterilized, acellular extracellular
matrix (ECM) compositions and methods for making such compositions.
As described herein, the disclosed extracellular matrix
compositions are formed by contemporaneously sterilizing and
decellularizing an isolated ECM material. More particularly, the
disclosed methods contemporaneously accomplish desired
sterilization and decellularization of the isolated ECM material
such that the native properties of the tissue composition are
maintained and the ECM material is rendered sterile and
acellular.
[0034] As further described herein, the disclosed methods make use
of rapid depressurization of the ECM material to decellularize the
ECM material such that it is acellular. This rapid depressurization
of the ECM material occurs at depressurization rates that are
significantly higher than the depressurization rates applied in
previously known methods. In addition to decellularizing the ECM
material as described herein, the rapid depressurization of the ECM
material also can be used to incorporate desired sterilants and
additives into the ECM material.
ECM Compositions
[0035] In exemplary aspects, a sterilized, acellular ECM
composition can comprise any known ECM component or material,
including, for example and without limitation, mucosal layers and
components, submucosal layers and components, muscularis layers and
components, and/or basement membrane layers and components. It is
contemplated that a disclosed sterilized, acellular ECM composition
can comprise an ECM material obtained from any mammalian tissue
source, including, for example and without limitation, stomach
tissue (e.g., stomach submucosa (SS)), small intestinal tissue
(e.g., small intestinal submucosa (SIS)), large intestinal tissue,
bladder tissue (e.g., urinary bladder submucosa (UBS)), liver
tissue (e.g., liver basement membrane (LBM)), heart tissue (e.g.,
pericardium), lung tissue, kidney tissue, pancreatic tissue,
prostate tissue, mesothelial tissue, fetal tissue, a placenta, a
ureter, veins, arteries, tissue surrounding the roots of developing
teeth, and tissue surrounding growing bone. It is further
contemplated that a disclosed sterilized, acellular ECM composition
can comprise an ECM material obtained from ECM components or
materials of one or more mammals including, for example and without
limitation, humans, cows, pigs, dogs, sheep, cats, horses, rodents,
and the like. Thus, it is contemplated that a disclosed sterilized,
acellular ECM composition can comprise ECM components or materials
from two or more of the same mammalian species, such as, for
example and without limitation, two or more cows, two or more pigs,
two or more dogs, or two or more sheep. It is further contemplated
that a disclosed sterilized, acellular ECM composition can comprise
ECM components or materials from two or more different mammalian
species, such as, for example and without limitation, a pig and a
cow, a pig and a dog, a pig and a sheep, or a cow and a sheep. It
is still further contemplated that a disclosed sterilized,
acellular ECM composition can comprise ECM components or materials
obtained from a first tissue source, such as, for example and
without limitation, SIS, from a first mammal, as well as ECM
components or materials obtained from a second tissue source, such
as, for example and without limitation, SS, from a second
mammal.
[0036] In various aspects, a disclosed sterilized, acellular ECM
composition can be produced in any suitable shape, including, for
example and without limitation, a substantially flat sheet, a
cylindrical tube, a substantially spherical structure, or a
multi-laminate structure. It is contemplated that a disclosed
sterilized, acellular ECM composition can also be produced in any
suitable form, including, for example and without limitation, a
solid, liquid, gel, particulate, emulsion, or suspension form. In
one exemplary aspect, it is contemplated that a disclosed
sterilized, acellular ECM composition can comprise an outer layer
of solid ECM material that encloses an inner layer of liquid,
particulate, emulsion, suspension, and/or gel ECM material.
[0037] In another exemplary aspect, it is contemplated that a
disclosed sterilized, acellular ECM composition can comprise one or
more types of particulate ECM materials that are suspended within
an ECM gel to form an ECM suspension. In this aspect, it is
contemplated that the particulates within a disclosed ECM
suspension can have a diameter ranging from about 5 .mu.m to about
300 .mu.m, with an average diameter ranging from about 90 .mu.m to
about 100 .mu.m. It is further contemplated that the percentage of
gel within a disclosed ECM suspension can range from about 5% to
about 50%, while the percentage of particulate within a disclosed
ECM suspension can range from about 50% to about 95%. Thus, it is
contemplated that the percentage of gel within a disclosed ECM
suspension can be about 10%, while the percentage of particulate
within the ECM suspension can be about 90%. It is further
contemplated that the percentage of gel within a disclosed ECM
suspension can be about 15%, while the percentage of particulate
within the ECM suspension can be about 85%. More preferably, the
percentage of gel within a disclosed ECM suspension can range from
about 20% to about 30%, while the percentage of particulate within
a disclosed ECM suspension can range from about 70% to about 80%.
Thus, in an exemplary aspect, the percentage of gel within a
disclosed ECM suspension can be about 20%, while the percentage of
particulate within the ECM suspension can be about 80%. In another
exemplary aspect, the percentage of gel within a disclosed ECM
suspension can be about 25%, while the percentage of particulate
within the ECM suspension can be about 75%. In an additional
exemplary aspect, the percentage of gel within a disclosed ECM
suspension can be about 30%, while the percentage of particulate
within the ECM suspension can be about 70%. Although the above
ranges refer to particular beginning point values and end point
values, it is contemplated that a disclosed ECM suspension can be
formed from gel percentages and particulate percentages falling
within any of the ranges disclosed above.
[0038] In a further aspect, it is contemplated that a disclosed ECM
suspension can comprise sterilized, decellularized ECM. In
exemplary aspects, the ECM gel of a disclosed ECM suspension can be
a hydrolyzed ECM. In these aspects, it is contemplated that the ECM
gel of a disclosed ECM suspension can comprise ECM that is greater
than about 50% hydrolyzed, more preferably, greater than about 70%
hydrolyzed, and, most preferably, greater than about 90%
hydrolyzed. In one exemplary aspect, the ECM gel of a disclosed ECM
suspension can comprise ECM that is about 100% hydrolyzed. It is
still further contemplated that the ECM components of the
suspension can comprise at least one of: glycoproteins, such as,
for example and without limitation, fibronectin and laminan;
glycosaminoglycans, such as, for example and without limitation,
heparan, hyaluronic acid, and chondroitin sulfate; and growth
factors, thereby providing additional bioavailability for native
cellular components. It is contemplated that the ECM components of
the suspension can provide a structural and biochemical
microenvironment that promotes cell growth and stem cell attraction
following implantation of a disclosed ECM suspension within a
subject. It is further contemplated that the ECM gel of a disclosed
ECM suspension can function as a bulking agent that preserves a
desired biomechanical environment until the cells of the subject
can begin producing their own ECM.
[0039] It is still further contemplated that the desired
biomechanical environment that is preserved by the ECM gel can
substantially correspond to a biomechanical environment in native
tissue. Thus, it is contemplated that the ECM gel of a disclosed
ECM suspension can have an elastic modulus that is substantially
equal to the elastic modulus of a target site within a subject. In
exemplary aspects, the elastic modulus of the ECM gel of a
disclosed ECM suspension can range from about 5 kPa to about 50
kPa, and, more preferably, from about 10 kPa to about 15 kPa.
[0040] In one non-limiting exemplary aspect, it is contemplated
that, when a disclosed ECM suspension is configured for injection
at a target site on or within the heart of a subject, the elastic
modulus of the ECM gel of the disclosed ECM suspension can be about
11.5 kPa, which is the elastic modulus of cardiac muscle. As used
herein, the term "on or within the heart" refers to locations that
are, for example and without limitation, on or within the
pericardium, epicardium, myocardium, endocardium, ventricles,
atria, aorta, pulmonary arteries, pulmonary veins, vena cavae, and
the like. In another aspect, it is further contemplated that a
disclosed ECM suspension can be injected at a target site on or
within the heart of the subject to therapeutically prevent or
reverse left ventricular wall negative remodeling that occurs
following acute myocardial infarction and/or chronic coronary heart
disease. As used herein, the term "negative remodeling" refers to
the detrimental and/or undesired changes in the heart that occur in
response to myocardial injury; such undesired changes include, for
example and without limitation, alterations in myocyte biology,
myocyte loss, extracellular matrix degradation, extracellular
matrix replacement fibrosis, alterations in left ventricular
chamber geometry, increased wall stress (afterload), afterload
mismatch, episodic subendocardial hypoperfusion, increased oxygen
utilization, sustained hemodynamic overloading, and worsening
activation of compensatory mechanisms. It is still further
contemplated that a disclosed ECM suspension can be injected at a
target site on or within the heart of the subject to
therapeutically treat heart failure.
[0041] In an exemplary aspect, it is contemplated that a disclosed
ECM suspension can be injected at a target site on or within the
heart of a subject, such as, for example and without limitation, on
or within the pericardium, epicardium, myocardium, endocardium,
ventricles, atria, aorta, and the like. Optionally, in one aspect,
a disclosed ECM suspension can be injected in a grid-like pattern.
In this aspect, it is contemplated that a disclosed ECM suspension
can be injected as a first series of spaced, substantially parallel
lines and a second series of spaced, substantially parallel lines
that are substantially perpendicular to the first series of spaced,
substantially parallel lines, thereby defining the grid-like
pattern.
[0042] In another aspect, it is contemplated that a disclosed ECM
suspension can be applied to a target site on or within the heart
of a subject to create a film of a disclosed ECM suspension having
a thickness ranging from about 0.1 mm to about 10 mm, more
preferably, from about 1 mm to about 5 mm, and, most preferably,
from about 2 mm to about 4 mm. In one exemplary aspect, it is
contemplated that a disclosed ECM suspension can be applied to a
target site on or within the heart of the subject to create a film
of the ECM suspension having a thickness of about 3 mm.
[0043] In a further exemplary aspect, it is contemplated that a
disclosed ECM suspension can be injected at a target site
positioned within the myocardium or scar tissue of the heart of a
subject. In this aspect, it is contemplated that a disclosed ECM
suspension can be injected into the myocardium or scar tissue
within the heart of the subject at a desired depth relative to an
outer surface of the pericardium. It is further contemplated that
the desired depth at which a disclosed ECM suspension is injected
can range from about 0.5 mm to about 5 mm, more preferably, from
about 1 mm to about 3 mm, and most preferably, from about 1.5 mm to
about 2.5 mm. In one exemplary aspect, it is contemplated that the
desired depth at which a disclosed ECM suspension is injected can
be about 2 mm. In this aspect, it is contemplated that the desired
depth at which a disclosed ECM suspension is injected can
correspond to a position proximate the junction between the
epicardium and the myocardium. It is further contemplated that the
desired depth at which a disclosed ECM suspension is injected can
correspond to a position proximate ischemic and/or inflamed and/or
injured heart tissue. In an exemplary aspect, it is contemplated
that the desired depth at which a disclosed ECM suspension is
injected can correspond to a position proximate necrotic and/or
infarcted myocardium.
[0044] In exemplary aspects, when a disclosed ECM suspension is to
be injected at a target site within the myocardium and/or one or
more chambers of the heart of a subject following the occurrence of
a myocardial infarction, it is contemplated that the ECM suspension
should be injected at the target site during one of two possible
time periods: prior to full onset of the inflammatory response of
the subject or after the inflammatory response of the subject has
decreased. In one aspect, when the ECM suspension is injected at
the target site prior to full onset of the inflammatory response of
the subject, it is contemplated that the ECM suspension should be
injected at the target site substantially immediately after
occurrence of the myocardial infarction up to the time of
therapeutic revascularization of the heart (using, for example, a
coronary artery bypass graft or a stent). In another aspect, when
the ECM suspension is injected at the target site after the
inflammatory response of the subject has decreased, it is
contemplated that the ECM suspension should be injected at the
target site after the acute phase of the myocardial infarction,
during which negatively remodeling and scar tissue formation occur.
In various aspects, it is contemplated that, following injection of
a disclosed ECM suspension on or within the heart of a subject, the
ECM suspension will not disperse but will instead attract stem
cells to the target site, thereby promoting desired positive
remodeling of the heart. As used herein, the term "positive
remodeling" refers to beneficial regeneration and/or restructuring
of damaged heart tissue; such positive remodeling promotes growth
of new cells while preserving the functionality of the heart and
preventing formation of scar tissue.
Sterilization and Decellularization of the ECM Compositions
[0045] Optionally, it is contemplated that the disclosed
extracellular matrix compositions can be sterilized using a known
sterilization system, such as, for example and without limitation,
the system described in U.S. Pat. No. 7,108,832, assigned to
NovaSterilis, Inc., which patent is expressly incorporated herein
by reference in its entirety. Thus, in some aspects, the system
used to perform the disclosed methods can comprise a standard
compressed storage cylinder and a standard air compressor used in
operative association with a booster (e.g., a Haskel Booster AGT
7/30). In other aspects, the air compressor and booster can be
replaced with a single compressor. In exemplary aspects, the
compressed storage cylinder can be configured to receive carbon
dioxide, and the booster can be a carbon dioxide booster.
[0046] The system can further comprise an inlet port, which allows
one or more additives contained in a reservoir to be added to a
reactor vessel through a valve and an additive line. As used
herein, the term "reactor vessel" refers to any container having an
interior space that is configured to receive an ECM material and
permit exposure of the ECM material to one or more sterilants and
additives, as disclosed herein. In exemplary aspects, the reactor
vessel can be, without limitation, a basket, a bucket, a barrel, a
box, a pouch, and other known containers. It is contemplated that
the reactor vessel can be re-sealable. In one aspect, it is
contemplated that the reactor vessel can be a syringe that is
filled with an ECM material. In an exemplary aspect, the reactor
vessel can be a pouch comprising Tyvek.RTM. packaging (E.I. du Pont
de Nemours and Company).
[0047] It is contemplated that a selected primary sterilant, such
as, for example and without limitation, carbon dioxide, can be
introduced to the reactor vessel from a header line via a valve and
a supply line. It is further contemplated that a filter, such as,
for example and without limitation, a 0.5 .mu.m filter, can be
provided in the supply line to prevent escape of material from the
vessel. In exemplary aspects, a pressure gauge can be provided
downstream of a shut-off valve in the header line to allow the
pressure to be visually monitored. A check valve can be provided in
the header line upstream of the valve to prevent reverse fluid flow
into the booster. In order to prevent an overpressure condition
existing in the header line, a pressure relief valve can optionally
be provided.
[0048] In one aspect, depressurization of the reactor vessel can be
accomplished using an outlet line and a valve in communication with
the reactor vessel. In this aspect, it is contemplated that the
depressurized fluid can exit the vessel via the supply line, be
filtered by a filter unit, and then be directed to a separator,
where filtered fluid, such as carbon dioxide, can be exhausted via
an exhaust line. It is further contemplated that valves can be
incorporated into the various lines of the apparatus to permit
fluid isolation of upstream components.
[0049] In one exemplary aspect, the reactor vessel can comprise
stainless steel, such as, for example and without limitation, 316
gauge stainless steel. In another exemplary aspect, the reactor
vessel can have a total internal volume sufficient to accommodate
the materials being sterilized, either on a laboratory or
commercial scale. For example, it is contemplated that the reactor
vessel can have a length of about 8 inches, an inner diameter of
about 2.5 inches, and an internal volume of about 600 mL. In
additional aspects, the reactor vessel can comprise a vibrator, a
temperature control unit, and a mechanical stirring system
comprising an impeller and a magnetic driver. In one optional
aspect, it is contemplated that the reactor vessel can contain a
basket comprising 316 gauge stainless steel. In this aspect, it is
contemplated that the basket can be configured to hold materials to
be sterilized while also protecting the impeller and directing the
primary sterilant in a predetermined manner.
[0050] It is contemplated that the reactor vessel can be operated
at a constant pressure or under continual pressurization and
depressurization (pressure cycling) conditions without material
losses due to splashing or turbulence, and without contamination of
pressure lines via back-diffusion. It is further contemplated that
the valves within the system can permit easy isolation and removal
of the reactor vessel from the other components of the system. In
one aspect, the top of the reactor vessel can be removed when
depressurized to allow access to the interior space of the reactor
vessel.
[0051] Optionally, the system can comprise a temperature control
unit that permits a user to adjustably control the temperature
within the reactor vessel.
[0052] In use, the disclosed apparatus can be employed in a method
of producing a sterilized, acellular ECM composition, such as
disclosed herein. However, it is understood that the disclosed
apparatus is merely exemplary, and that any apparatus capable of
performing the disclosed method steps can be employed to produce
the sterilized, acellular ECM composition. Thus, the claimed method
is in no way limited to a particular apparatus.
[0053] It is contemplated that significant reductions in
colony-forming units (CFUs) can be achieved in accordance with the
disclosed methods by subjecting an isolated ECM material to
sterilization temperature and pressure conditions using a primary
sterilant. Optionally, it is contemplated that the primary
sterilant can be combined with one or more secondary sterilants to
achieve desired sterilization. Optionally, it is further
contemplated that selected additives can be incorporated into an
ECM material to impart desired characteristics to the resulting ECM
composition. It is still further contemplated that the disclosed
methods can be employed to produce sterilized, acellular ECM
compositions for implantation within the body of a subject.
[0054] As described herein, the disclosed methods make use of rapid
depressurization of an isolated ECM material to render the ECM
material acellular. This rapid depressurization of the ECM material
occurs at depressurization rates that are significantly higher than
the depressurization rates applied in previously known methods. In
addition to rendering acellular the ECM material as described
herein, the rapid depressurization of the ECM material also can be
used to enhance the incorporation of desired sterilants and
additives into the ECM material. Further, it is contemplated that
the rapid depressurization of the ECM material can render the ECM
material acellular while also improving retention of native growth
factors, as compared to previously known decellularization methods.
Still further, it is contemplated that the rapid depressurization
of the ECM material can be used to improve retention of the tensile
strength of the ECM material, as compared to previously known
decellularization methods.
[0055] The disclosed methods not only do not significantly weaken
the mechanical strength and bioptric properties of the ECM
compositions, but also the methods are more effective in
decellularizing the ECM compositions and in enhancing the
incorporation of various additives into the ECM compositions. Thus,
the disclosed sterilization and decellularization methods provide
ECM compositions that are more decellularized and have a greater
capacity to incorporate and then deliver more additives than ECM
compositions known in the art. Moreover, the disclosed
sterilization and decellularization methods provide ECM
compositions that have greater amounts and/or concentrations of
retained native growth factors and that have greater tensile
strength than sterilized and decellularized ECM compositions known
in the art.
[0056] In exemplary aspects, the primary sterilant can be carbon
dioxide at or near its supercritical pressure and temperature
conditions. However, it is contemplated that any conventional
sterilant, including, for example, gas, liquid, or powder
sterilants that will not interfere with the native properties of
the ECM material, can be used as the primary sterilant.
[0057] In one exemplary aspect, the disclosed sterilization process
can be practiced using carbon dioxide as a primary sterilant at
pressures ranging from about 1,000 to about 3,500 psi and at
temperatures ranging from about 25.degree. C. to about 60.degree.
C. More preferably, when supercritical carbon dioxide is used, it
is contemplated that the sterilization process can use carbon
dioxide as a primary sterilant at pressures at or above 1,071 psi
and at temperatures at or above 31.1.degree. C. In this aspect, the
ECM material to be sterilized can be subjected to carbon dioxide at
or near such pressure and temperature conditions for times ranging
from about 10 minutes to about 24 hours, more preferably from about
15 minutes to about 18 hours, and most preferably, from about 20
minutes to about 12 hours. Preferably, the carbon dioxide employed
in the disclosed systems and methods can be pure or, alternatively,
contain only trace amounts of other gases that do not impair the
sterilization properties of the carbon dioxide. For ease of further
discussion below, the term "supercritical carbon dioxide" will be
used, but it will be understood that such a term is non-limiting in
that carbon dioxide within the pressure and temperature ranges as
noted above can be employed satisfactorily in the practice of the
disclosed methods. Within the disclosed pressure and temperature
ranges, it is contemplated that the carbon dioxide can be presented
to the ECM material in a gas, liquid, fluid or plasma form.
[0058] The secondary sterilants employed in the disclosed methods
can, in some aspects, include chemical sterilants, such as, for
example and without limitation, peroxides and/or carboxylic acids.
Preferred carboxylic acids include alkanecarboxylic acids and/or
alkanepercarboxylic acids, each of which can optionally be
substituted at the alpha carbon with one or more
electron-withdrawing substituents, such as halogen, oxygen and
nitrogen groups. Exemplary species of chemical sterilants employed
in the practice of the disclosed methods include, for example and
without limitation, hydrogen peroxide (H.sub.2O.sub.2), acetic acid
(AcA), peracetic acid (PAA), trifluoroacetic acid (TFA), and
mixtures thereof. In one exemplary aspect, the chemical sterilants
can include Sporeclenz.RTM. sterilant, which is a mixture
comprising acetic acid, hydrogen peroxide, and peracetic acid.
[0059] It is contemplated that the secondary sterilants can be
employed in a sterilization-enhancing effective amount of at least
about 0.001 vol. % and greater, based on the total volume of the
primary sterilant. It is further contemplated that the amount of
secondary sterilant can be dependent upon the particular secondary
sterilant that is employed. Thus, for example, it is contemplated
that peracetic acid can be present in relatively small amounts of
about 0.005 vol. % and greater, while acetic acid can be employed
in amounts of about 1.0 vol. % and greater. Thus, it is
contemplated that the concentration of the secondary sterilants can
range from about 0.001 vol. % to about 2.0 vol. % and can typically
be used as disclosed herein to achieve a sterilization-enhancing
effect in combination with the disclosed primary sterilants, such
as, for example and without limitation, supercritical carbon
dioxide.
[0060] In one aspect, the method of producing a sterilized,
acellular ECM composition can comprise harvesting a selected tissue
from a mammal and rinsing the selected tissue in sterile saline or
other biocompatible liquid, including, for example and without
limitation, Ringer's solution or a balanced biological salt
solution. In this aspect, the selected tissue can be, for example
and without limitation, stomach tissue (e.g., stomach submucosa
(SS)), small intestinal tissue (e.g., small intestinal submucosa
(SIS)), large intestinal tissue, bladder tissue (e.g., urinary
bladder submucosa (UBS)), liver tissue (e.g., liver basement
membrane (LBM)), heart tissue (e.g., pericardium, epicardium,
endocardium, myocardium), lung tissue, kidney tissue, pancreatic
tissue, prostate tissue, mesothelial tissue, fetal tissue, a
placenta, a ureter, veins, arteries, heart valves with or without
their attached vessels, tissue surrounding the roots of developing
teeth, and tissue surrounding growing bone. In another aspect, the
method can comprise freezing the selected tissue for a period
ranging from about 12 to about 36 hours, more preferably, from
about 18 to about 30 hours, and most preferably, from about 22 to
about 26 hours. For example, it is contemplated that the period
during which the selected tissue is frozen can be 12 hours, 13
hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours,
20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26
hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours,
33 hours, 34 hours, 35 hours, 36 hours, and any other period of
time falling between the preceding values. In an additional aspect,
the method can comprise thawing the selected tissue in cold
hypotonic tris buffer. Optionally, in this aspect, the method can
comprise thawing the selected tissue in cold hypotonic tris buffer
on ice with 5 mM ethylenediaminetetraacetic acid (EDTA). In
exemplary aspects, it is contemplated that the steps of freezing
and thawing the selected tissue can be cyclically repeated up to
six times.
[0061] In another aspect, the method can comprise isolating an ECM
material from the selected tissue. In this aspect, the ECM material
can be any material comprising known extracellular matrix
components, including, for example and without limitation, stomach
tissue (e.g., stomach submucosa (SS)), small intestinal tissue
(e.g., small intestinal submucosa (SIS)), large intestinal tissue,
bladder tissue (e.g., urinary bladder submucosa (UBS)), liver
tissue (e.g., liver basement membrane (LBM)), heart tissue (e.g.,
pericardium, epicardium, endocardium, myocardium), lung tissue,
kidney tissue, pancreatic tissue, prostate tissue, mesothelial
tissue, fetal tissue, a placenta, a ureter, veins, arteries, heart
valves with or without their attached vessels, tissue surrounding
the roots of developing teeth, and tissue surrounding growing bone.
In one exemplary, non-limiting aspect, the step of isolating an ECM
material can comprise isolating SIS from a mammalian tissue source.
In this aspect, the method can comprise: incising a wall of a small
intestine along a path that is substantially parallel to the
longitudinal axis of the small intestine; opening the small
intestine along the path of the incision such that the small
intestine lies flat on a surface; rinsing the small intestine with
sterile saline or other biocompatible fluid; mechanically stripping
the SIS of the small intestine from the surrounding smooth muscle
and serosal layers and from the tunica mucosa, leaving essentially
the submucosal and basement membrane layers. However, it is
contemplated that the ECM material can be isolated using any
conventional technique, including those described in: U.S. Pat. No.
4,902,508; U.S. Pat. No. 5,275,826; U.S. Pat. No. 5,281,422; U.S.
Pat. No. 5,554,389; U.S. Pat. No. 6,579,538; U.S. Pat. No.
6,933,326; U.S. Pat. No. 7,033,611; Voytik-Harbin et al.,
"Identification of Extractable Growth Factors from Small Intestinal
Submucosa," J. Cell. Biochem., Vol. 67, pp. 478-491 (1997); Hodde
et al., "Virus Safety of a Porcine-Derived Medical Device:
Evaluation of a Viral Inactivation Method," Biotech. & Bioeng.,
Vol. 79, No. 2, pp. 211-216 (2001); Badylak et al., "The
Extracellular Matrix as a Scaffold for Tissue Reconstruction," Cell
& Developmental Biology, Vol. 13, pp. 377-383 (2002); Robinson
et al., "Extracelular Matrix Scaffold for Cardiac Repair,"
Circulation, Vol. 112, pp. I-135-I-143 (2005); Hodde et al.,
"Effects of Sterilization on an Extracellular Matrix Scaffold: Part
I. Composition and Matrix Architecture," J. Mater. Sci.: Mater.
Med., Vol. 18, pp. 537-543 (2007); and Hodde et al., "Effects of
Sterilization on an Extracellular Matrix Scaffold: Part II.
Bioactivity and Matrix Interaction," J. Mater. Sci.: Mater. Med.,
Vol. 18, pp. 545-550 (2007), each of which is expressly
incorporated herein by reference in its entirety.
[0062] In an additional aspect, the method can comprise incubating
the isolated ECM material for 24 to 48 hours in 0.5-1% Triton
X-100/0.5-1% Deoxycholic acid with 5 mM EDTA in Dulbecco's
Phosphate Buffered Saline (DPBS) (Lonza Walkersville, Inc.). In
this aspect, it is contemplated that flat or sheet-like ECM
materials, such as stomach submucosa (SS), small intestinal
submucosa (SIS), and bladder submucosa (UBS), can be incubated in a
stretched configuration. It is further contemplated that ECM
material conduits or other lumenal ECM materials, such as ureters,
arteries, veins, and tubular SIS, can be perfused with the various
disclosed solutions through soaking and by use of a peristaltic
pump.
[0063] In a further aspect, after incubation, the method can
comprise rinsing the ECM material with DPBS. In this aspect, it is
contemplated that the step of rinsing the ECM material can comprise
rinsing the ECM material up to six times, including one, two,
three, four, five, or six times, with each rinse lasting for about
thirty minutes. In an exemplary aspect, it is contemplated that the
step of rinsing the ECM material can comprise rinsing the ECM
material three times, with each rinse lasting for about thirty
minutes.
[0064] Optionally, in exemplary aspects, the method can further
comprise a second incubation procedure. In these aspects, the
second incubation procedure can comprise incubating the ECM
material in isotonic tris buffer containing 10-50 .mu.g/mL of
RNAase/0.2-0.5 .mu.g/mL DNAase with 5 mM EDTA. It is contemplated
that the step of incubating the ECM material in isotonic tris
buffer can be performed at a temperature of about 37.degree. C.,
substantially corresponding to the temperature of a human body. It
is further contemplated that the step of incubating the ECM
material in isotonic tris buffer can be performed for a period
ranging from about 30 minutes to about 24 hours, more preferably,
from about 1 hour to about 18 hours, and most preferably, from
about 2 hours to about 12 hours. In an additional aspect, the
second incubation procedure can further comprise rinsing the ECM
material with DPBS. In this aspect, it is contemplated that the
step of rinsing the ECM material can comprise rinsing the ECM
material three times, with each rinse lasting for about thirty
minutes.
[0065] In yet another aspect, whether or not the second incubation
procedure is performed, the method can comprise storing the ECM
material at a temperature ranging from about 1.degree. C. to about
10.degree. C., more preferably, from about 2.degree. C. to about
6.degree. C., and, most preferably, from about 3.degree. C. to
about 5.degree. C. In an exemplary aspect, the ECM material can be
stored at 4.degree. C.
[0066] In an additional aspect, the method can comprise introducing
the ECM material into the interior space of the reactor vessel.
Optionally, in this aspect, one or more secondary sterilants from
the reservoir can be added into the interior space of the reactor
vessel along with the ECM material. In these aspects, it is
contemplated that the one or more secondary sterilants from the
reservoir can be added into the interior space of the reactor
vessel before, after, or contemporaneously with the ECM material.
It is further contemplated that the temperature control unit can be
selectively adjusted to produce a desired temperature within the
interior space of the reactor vessel. In a further aspect, the
method can comprise equilibrating the pressure within the reactor
vessel and the pressure within the storage cylinder. For example,
in this aspect, it is contemplated that the pressure within the
reactor vessel and the pressure within the storage cylinder can be
substantially equal to atmospheric pressure. In yet another aspect,
after equilibration of the pressures within the apparatus, the
method can comprise operating the magnetic driver to activate the
impeller of the reactor vessel. In still a further aspect, the
method can comprise selectively introducing the primary sterilant
from the storage cylinder into the reactor vessel until a desired
pressure within the reactor vessel is achieved. In this aspect, it
is contemplated that the step of selectively introducing the
primary sterilant into the reactor vessel can comprise selectively
activating the air compressor and the booster to increase flow of
the primary sterilant into the reactor vessel. In exemplary
aspects, the air compressor and booster can be activated to subject
the ECM material to supercritical pressures and temperatures, such
as, for example and without limitation, the pressures and
temperatures necessary to produce supercritical carbon dioxide, for
a time period ranging from about 20 minutes to about 60
minutes.
[0067] In a further aspect, the method can comprise rapidly
depressurizing the reactor vessel. In this aspect, a predetermined
amount of primary sterilant, such as, for example and without
limitation, supercritical carbon dioxide, can be released from the
reactor vessel through the depressurization line. It is
contemplated that the primary sterilant can be released from the
reactor vessel through opening of the valve coupled to the reactor
vessel to thereby rapidly reduce the pressure within the reactor
vessel. As used herein, the term "rapid depressurization" refers to
depressurization of the reactor vessel at a rate greater than or
equal to 400 psi/min. For example, it is contemplated that the
reactor vessel can be depressurized at a depressurization rate
ranging from about 2.9 MPa/min. to about 18.0 MPa/min. (about 400
psi/min. to about 2,600 psi/min.), more preferably from about 5.0
MPa/min. to about 10.0 MPa/min. (700 psi/min. to about 1,500
psi/min.), and, most preferably, from about 7.0 MPa/min. to about
8.0 MPa/min. (about 1,000 psi/min. to about 1,200 psi/min.). Thus,
these rapid depressurizations are significantly greater than the
300 psi/min. depressurization rate disclosed in U.S. Pat. No.
7,108,832. Without being bound by any particular theory, it is
believed that the disclosed rapid depressurization rates increase
the level of decellularization achieved in the ECM material. For
example, the rapid depressurization of a disclosed ECM material can
lead to levels of decellularization in the ECM material of greater
than about 96%, including 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%,
96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%,
97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%,
98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and 100.0%.
[0068] In exemplary aspects, the method can further comprise the
step of incorporating one or more additives into the ECM material.
In these aspects, it is contemplated that the one or more additives
can be provided in either a powder or a liquid form. In one
optional aspect, the step of incorporating the one or more
additives can comprise introducing the one or more additives into
the reactor vessel during the step of rapidly depressurizing the
reactor vessel. In this aspect, it is contemplated that the
introduction of the one or more additives can be characterized as a
conventional foaming process. In another optional aspect, the step
of incorporating the one or more additives can comprise introducing
the one or more additives into the reactor vessel after the step of
rapidly depressurizing the reactor vessel. In this aspect, it is
contemplated that the one or more additives can be added to the ECM
material after the rapid depressurization of the reactor vessel has
caused the ECM material to swell and/or expand, thereby permitting
improved penetration of the additives into the ECM material. It is
further contemplated that, in an exemplary aspect, the one or more
additives can be added to the ECM material within about thirty
minutes after the rapid depressurization of the reactor vessel. In
a further optional aspect, the step of incorporating the one or
more additives can comprise introducing the one or more additives
into the reactor vessel both during and after the step of rapidly
depressurizing the reactor vessel. In this aspect, it is
contemplated that the one or more additives can be released into
the reactor vessel in both a quick manner and a slow, extended
manner. In still a further optional aspect, the step of
incorporating the one or more additives can comprise introducing
the one or more additives into the reactor vessel before the step
of rapidly depressurizing the reactor vessel.
[0069] The disclosed additives can be incorporated into the ECM
material to impart selected properties to the resulting sterilized,
acellular ECM composition. Thus, it is contemplated that the one or
more additives can be selected to replace or supplement components
of the ECM material that are lost during processing of the ECM
material as described herein. For example, and as described below,
the one or more additives can comprise growth factors, cytokines,
proteoglycans, glycosaminoglycans (GAGs), proteins, peptides,
nucleic acids, small molecules, drugs, or cells. It is further
contemplated that the one or more additives can be selected to
incorporate non-native components into the ECM material. For
example, the one or more additives can comprise, for example and
without limitation, growth factors for recruiting stem cells,
angiogenic cytokines, and anti-inflammatory cytokines. It is still
further contemplated that the one or more additives can be
pharmaceutical agents, such as statins, corticosteroids,
non-steroidal anti-inflammatory drugs, anti-inflammatory compounds,
anti-arrhythmic agents, and the like. It is still further
contemplated that the one or more additives can be nanoparticles,
such as, for example and without limitation, silver nanoparticles,
gold nanoparticles, platinum nanoparticles, iridium nanoparticles,
rhodium nanoparticles, palladium nanoparticles, copper
nanoparticles, zinc nanoparticles, and other metallic
nanoparticles. It is still further contemplated that the one or
more additives can be metallic compounds. In one exemplary aspect,
the one or more additives can be selected to pharmaceutically
suppress the immune response of a subject following implantation of
the resulting ECM composition into the body of a subject.
[0070] In one aspect, the one or more additives can comprise one or
more growth factors, including, for example and without limitation,
transforming growth factor-.beta.-1, -2, or -3 (TGF-.beta.-1, -2,
or -3), fibroblast growth factor-2 (FGF-2), also known as basic
fibroblast growth factor (bFGF), vascular endothelial growth factor
(VEGF), placental growth factor (PGF), connective tissue growth
factor (CTGF), hepatocyte growth factor (HGF), Insulin-like growth
factor (IGF), macrophage colony stimulating factor (M-CSF),
platelet derived growth factor (PDGF), epidermal growth factor
(EGF), and transforming growth factor-.alpha. (TGF-.alpha.).
[0071] In another aspect, the one or more additives can comprise
one or more cytokines, including, for example and without
limitation, stem cell factor (SCF), stromal cell-derived factor-1
(SDF-1), granulocyte macrophage colony-stimulating factor (GM-CSF),
interferon gamma (IFN-gamma), Interleukin-3, Interleukin-4,
Interleukin-10, Interleukin-13, Leukemia inhibitory factor (LIF),
amphiregulin, thrombospondin 1, thrombospondin 2, thrombospondin 3,
thrombospondin 4, thrombospondin 5, and angiotensin converting
enzyme (ACE).
[0072] In an additional aspect, the one or more additives can
comprise one or more proteoglycans, including, for example and
without limitation, heparan sulfate proteoglycans, betaglycan,
syndecan, decorin, aggrecan, biglycan, fibromodulin, keratocan,
lumican, epiphycan, perlecan, agrin, testican, syndecan, glypican,
serglycin, selectin, lectican, versican, neurocan, and
brevican.
[0073] In a further aspect, the one or more additives can comprise
one or more glycosaminoglycans, including, for example and without
limitation, heparan sulfate, hyaluronic acid, heparin, chondroitin
sulfate B (dermatan sulfate), and chondroitin sulfate A.
[0074] In still a further aspect, the one or more additives can
comprise one or more proteins, peptides, or nucleic acids,
including, for example and without limitation, collagens, elastin,
vitronectin, versican, laminin, fibronectin, fibrillin-1,
fibrillin-2, plasminogen, small leucine-rich proteins, cell-surface
associated protein, cell adhesion molecules (CAMs), a matrikine, a
matrix metalloproteinase (MMP), a cadherin, an immunoglobin, a
multiplexin, cytoplasmic domain-44 (CD-44), amyloid precursor
protein, tenascin, nidogen/entactin, fibulin I, fibulin II,
integrins, transmembrane molecules, and osteopontin.
[0075] In yet another aspect, the one or more additives can
comprise one or more pharmaceutical agents, including, for example
and without limitation, statin drugs, for example, cerevastatin,
atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,
pravastatin, rosuvastatin, and simvastatin, corticosteroids,
non-steroidal anti-inflammatory drugs, anti-inflammatory compounds,
anti-arrhythmic agents, antimicrobials, antibiotics, and the
like.
[0076] In exemplary aspects, the steps of introducing the one or
more additives into the reactor vessel can comprise opening the
valve to allow the one or more additives to flow from the reservoir
into the inlet port. Prior to pressurization, it is contemplated
that the one or more additives can be introduced directly into the
reactor vessel prior to sealing and/or via the inlet port.
[0077] It is contemplated that the disclosed rapid depressurization
and repressurization of the reactor vessel, with or without the
addition of the one or more additives, can be repeated for any
desired number of cycles. It is further contemplated that the
cycles of depressurization and repressurization, as well as the
introduction of the primary sterilants and/or secondary sterilants
and/or additives, can be automatically controlled via a controller
that is configured to selectively open and/or close the various
valves of the system to achieve desired pressure conditions and
cycles.
[0078] In some aspects, the disclosed methods can further comprise
the step of agitating the contents of the reactor vessel. In these
aspects, it is contemplated that the step of agitating the contents
of the reactor vessel can comprise periodically agitating the
contents of the reactor vessel using a vibrator. It is further
contemplated that the agitation of the reactor vessel can be
intermittent, continual, or continuous. In exemplary aspects, the
step of agitating the contents of the reactor vessel can occur
during the step of introducing the primary sterilant into the
reactor vessel. It is contemplated that the agitation of the
contents of the reactor vessel can enhance the mass transfer of the
sterilants and/or additives by eliminating voids in the fluids
within the reactor vessel to provide for more complete contact
between the ECM material and the sterilants and/or additives. It is
further contemplated that the step of agitating the contents of the
reactor vessel can comprise selectively adjusting the intensity and
duration of agitation so as to optimize sterilization times,
temperatures, and pressurization/depressurization cycles.
[0079] In a further aspect, after the sterilization and
decellularization of the ECM material is complete, the method can
further comprise depressurizing the reactor vessel and deactivating
the magnetic drive so as to cease movement of the stirring
impeller. Finally, the method can comprise the step of removing the
resulting sterilized, acellular ECM composition through the top of
the reactor vessel.
[0080] It is contemplated that the duration of the disclosed steps,
as well as the temperatures and pressures associated with the
disclosed steps, can be selectively varied to account for
variations in the characteristics of the ECM material. For example,
when the ECM material is a multi-laminate structure, has an
increased thickness, or is positioned within a syringe, it is
contemplated that the duration of the disclosed steps can be
increased.
[0081] In one optional aspect, in order to make the sterilized,
acellular ECM composition into a particulate form, the method can
comprise cutting the ECM composition into pieces having desired
lengths. In another aspect, the method can optionally comprise
freeze-drying the pieces of the ECM composition. In an additional
aspect, the method can optionally comprise grinding the frozen,
hydrated pieces of the ECM composition and then passing the pieces
of the ECM composition through a sizer screen until ECM particulate
of a desired size is isolated. In a further optional aspect, the
method can comprise rehydrating the ECM particulate with sterile
saline or other sterile, biocompatible fluid to form an ECM
suspension, as described herein.
Methods of Enhancing the Incorporation of an Additive into an ECM
Material
[0082] In exemplary aspects, a method for enhancing the
incorporation of an additive into an extracellular matrix (ECM)
material can comprise: positioning an ECM material within an
interior space of a reactor vessel; introducing carbon dioxide into
the interior space of the reactor vessel at supercritical pressure
and temperature conditions, thereby sterilizing the ECM material;
rapidly depressurizing the interior space of the reactor vessel at
a depressurization rate sufficient to render the ECM material
acellular and to increase the capacity of the ECM material for
incorporation of an additive; and introducing one or more additives
into the interior space of the reactor vessel, whereby at least a
portion of each additive of the one or more additives is
incorporated into the sterilized and acellular ECM material.
[0083] It is contemplated that the depressurization rate can range
from about 400 psi/minute to about 2,600 psi/minute, more
preferably, from about 700 psi/minute to about 1,500 psi/minute
and, most preferably, from about 1,000 psi/minute to about 1,200
psi/minute. It is further contemplated that the method for
enhancing the incorporation of an additive into the ECM material
can further comprise introducing at least one secondary sterilant
into the interior space of the reactor vessel. It is still further
contemplated that the one or more additives can comprise at least
one growth factor. It is still further contemplated that the one or
more additives can comprise at least one cytokine. It is still
further contemplated that the one or more additives can comprise at
least one proteoglycan. It is still further contemplated that the
one or more additives can comprise at least one glycosaminoglycan
(GAG). It is still further contemplated that the one or more
additives can comprise at least one of a protein, a peptide, and a
nucleic acid. It is still further contemplated that the one or more
additives can comprise at least one pharmaceutical agent. It is
still further contemplated that the one or more additives can
comprise nanoparticles.
[0084] Optionally, it is contemplated that the step of introducing
the one or more additives into the interior space of the reactor
vessel is performed contemporaneously with the step of rapidly
depressurizing the interior space of the reactor vessel.
[0085] It is further contemplated that a sterilized, acellular ECM
composition can be produced using the disclosed method for
enhancing the incorporation of an additive into an ECM material. In
exemplary aspects, the sterilized, acellular ECM composition can
comprise at least one tissue, and each tissue of the at least one
tissue can be from a respective tissue source. In these aspects, it
is contemplated that the tissue source of each tissue of the at
least one tissue can be selected from the group consisting of small
intestinal submucosa, stomach submucosa, large intestinal tissue,
urinary bladder submucosa, liver basement membrane, pericardium,
epicardium, endocardium, myocardium, lung tissue, kidney tissue,
pancreatic tissue, prostate tissue, mesothelial tissue, fetal
tissue, a placenta, a ureter, veins, arteries, heart valves with or
without their attached vessels, tissue surrounding the roots of
developing teeth, and tissue surrounding growing bone. In one
aspect, the sterilized, acellular ECM composition can be at least
about 96% decellularized, as set forth herein.
EXAMPLES
[0086] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Example 1
[0087] In exemplary applications of the disclosed sterilization and
decellularization methods, selected tissues were harvested and
rinsed in sterile saline. The selected tissues were then frozen for
24 hours. The frozen tissues were thawed in cold hypotonic tris
buffer on ice with 5 mM ethylenediaminetetraacetic acid (EDTA). An
extracellular matrix material was then isolated from each selected
tissue, as described herein.
[0088] The isolated extracellular matrix materials were incubated
for 24 to 48 hours in 0.5-1% Triton X-100/0.5-1% Deoxycholic acid
with 5 mM EDTA in Dulbecco's Phosphate Buffered Saline (DPBS)
(Lonza Walkersville, Inc.). Flat extracellular matrix materials,
such as stomach submucosa (SS), small intestinal submucosa (SIS),
and bladder submucosa (UBS), were incubated in a stretched
configuration. Tubular extracellular matrix materials, such as
ureters, arteries, veins, and tubular SIS, were perfused with the
solutions through soaking and by use of a peristaltic pump.
[0089] After incubation, each extracellular matrix material was
rinsed three times with DPBS. Each rinsing with DPBS lasted 30
minutes. Some extracellular matrix materials were then incubated
for 2 to 12 hours at 37.degree. C. in isotonic tris buffer
containing 10-50 .mu.g/mL of RNAse/0.2-0.5 .mu.g/mL DNAse with 5 mM
EDTA. Following this incubation step, the extracellular matrix
materials were again rinsed three times with DPBS. Each rinsing
with DPBS lasted 30 minutes. The extracellular matrix materials
were stored at 4.degree. C.
[0090] Within 48 hours of storage, the extracellular matrix
materials were processed in supercritical carbon dioxide as
disclosed herein for 20-60 minutes at temperatures at or greater
than 31.1.degree. C. and pressures at or greater than 1,071 psi.
After this sterilization step, the extracellular matrix materials
were rapidly depressurized at a rate of 2.7 MPa/10 sec. (391.6
psi/10 sec.) for a minute and 19 seconds. During this time, the
pressure applied to the extracellular matrix materials rapidly
decreased from 9.9 MPa to 0.69 MPa.
[0091] The extracellular matrix materials were then processed in
supercritical carbon dioxide and peracetic acid (PAA) as disclosed
herein for 30 minutes to 6 hours to achieve terminal sterilization.
In this processing step, the pressure applied to the extracellular
matrix materials was increased to 9.9 MPa. The resulting
sterilized, acellular extracellular matrix materials were then
packaged in Tyvek.RTM. (E.I. du Pont de Nemours & Company)
pouches that were sealed within plastic pouches to prevent fluid
leakage.
[0092] Table 1 summarizes the sterilization and decellularization
of porcine ureter, bovine pericardium, and porcine mesothelium.
TABLE-US-00001 TABLE 1 TX-100/ RNAse/ Triton Deoxy DNAse
Supercritical X-100 Deoxycholic incuba- incuba- CO.sub.2/PAA
Material Conc. Acid Conc. tion tion time Porcine 0.5% 0.5% 24 hours
2 hours 120 minutes ureters Bovine 0.5% 0.5% 24 hours 2 hours 180
minutes pericardium Porcine 0.5% 0.5% 24 hours 2 hours 120 minutes
mesothelium
Example 2
[0093] The DNA content of ECM material samples was measured as an
indicator of decellularization of the respective ECM material
samples using various sterilization and decellularization
techniques. The measured DNA content was evaluated with a pico
green assay in which DNA was labeled with a fluorescent label that
was detected with a spectrophotometer. The measured DNA content was
normalized by the dry weight of the samples. DNA content was
measured and evaluated for the following treatment groups: (1)
Lyophilized, non-sterile SIS; (2) Ethylene Oxide (EtO)-sterilized
SIS; (3) Lyophilized, non-sterile SIS that was sterilized through a
60 minute treatment with PAA and supercritical CO.sub.2, as
disclosed herein; (4) Lyophilized, non-sterile SIS that was
sterilized through a 20 minute treatment with PAA and supercritical
CO.sub.2, as disclosed herein; and (5) Raw, unprocessed SIS.
[0094] FIG. 1 shows the total DNA content for the respective
samples, as normalized by dry weight. FIG. 2 shows the percent of
DNA that was removed from each respective sample, as compared to
raw, unprocessed SIS. These results indicated that by sterilizing
the non-sterile SIS using a 60 minute treatment with PAA and
supercritical CO.sub.2, as disclosed herein, over 96% of the DNA
found in raw SIS was removed, as compared to only 94% when the SIS
was sterilized by EtO and only 93% when the SIS was not sterilized
by any method.
Example 3
[0095] Ureters were processed with a gentle detergent (0.5% Triton
X-100/0.5% Sodium Deoxycholate in 5 mM EDTA in DPBS) for 24 hours
and then rinsed three times in DPBS as disclosed herein. After this
pretreatment, the ureters were decellularized and sterilized using
rapid depressurization and treatment with PAA and supercritical
CO.sub.2, as disclosed herein. Hematoxylin and Eosin (H&E)
Stains were prepared for one sample ureter at the following stages
of treatment: (A) native ureter; (B) pretreated ureter; and (C)
pretreated ureter with rapid depressurization and treatment with
PAA and supercritical CO.sub.2, as disclosed herein. These stains
indicated that DNA content was significantly reduced with rapid
depressurization.
Example 4
[0096] The growth factor content of ECM material samples was
measured. Enzyme-linked immunosorbent (ELISA) assays were performed
on the ECM material samples to quantify the content of bFGF and the
active form of TGF-.beta. in each respective sample. The following
treatment groups were evaluated: (1) Lyophilized, non-sterile SIS;
(2) Ethylene Oxide (EtO)-sterilized SIS; (3) Lyophilized,
non-sterile SIS that was sterilized through a 60 minute treatment
with PAA and supercritical CO.sub.2, as disclosed herein; (4)
Lyophilized, non-sterile SIS that was sterilized through a 20
minute treatment with PAA and supercritical CO.sub.2, as disclosed
herein; and (5) Raw, unprocessed SIS. The bFGF content and
TGF-.beta. content measurements were normalized by dry weight of
each respective sample. These results are shown in FIGS. 3 and 4.
These results indicated that the concentration of both growth
factors was reduced by exposure to EtO. However, the concentration
of the growth factors was not affected by sterilization with PAA
and supercritical CO.sub.2.
Example 5
[0097] Using the methods disclosed herein, supercritical CO.sub.2
was used as a primary sterilant and as a carrier for adding bFGF
into SIS sheets. First, the respective SIS sheets were placed into
Tyvek.RTM. pouches along with varying amounts of bFGF. The pouches
were exposed to supercritical CO.sub.2 for 60 minutes at 9.6 MPa.
The pouches were rapidly depressurized at a rate of 7.20 MPa/min.
Samples were directly processed in 16 mL PAA in supercritical
CO.sub.2 for 20 minutes. The following treatment groups were
evaluated: (1) No bFGF added; (2) 5 .mu.L bFGF added; and (3) 15
.mu.L bFGF added. Each .mu.L of bFGF contained 0.1 .mu.g of bFGF.
Thus, since each SIS sheet weighed approximately 0.5 g, the maximum
concentrations of bFGF for the 5 .mu.L and 15 .mu.L groups were
about 4170 pg/mg dry weight and about 12,500 pg/mg dry weight,
respectively. The bFGF content for these groups is shown in FIG. 5,
as measured with respect to the dry weight of the respective
samples. These results indicated that the measured concentrations
of bFGF did not reach the maximum concentrations and that the
sample to which 15 .mu.L of bFGF was added did not have a measured
concentration of bFGF that was three times greater than the
measured concentration of bFGF in the sample to which 5 .mu.L of
bFGF was added.
Example 6
[0098] The tensile strengths of two-ply SIS samples were measured.
The following treatment groups were evaluated: (1) EtO Treatment;
(2) PAA/supercritical CO.sub.2 treatment for 20 minutes; (3)
PAA/supercritical CO.sub.2 treatment for 60 minutes; and (4)
PAA/supercritical CO.sub.2 treatment for 120 minutes. The tensile
strength test results are shown in FIG. 6. These results indicated
that the SIS samples that were processed with PAA/supercritical
CO.sub.2 for 20 or 120 minutes, as disclosed herein, were
significantly stronger than the SIS samples that were processed
with EtO.
Example 7
[0099] Rapid depressurization was used following gentle detergent
soaks or perfusion of the ECM materials listed in Table 2 (below)
at the noted concentrations and for the noted time periods. Tissues
were harvested and rinsed in saline. The tissues were frozen for at
least 24 hours. The tissues were thawed in cold hypotonic tris
buffer on ice with 5 mM EDTA. The ECM of interest was isolated. For
flat tissues (e.g., stomach submucosa, small intestine submucosa,
and bladder submucosa), the tissue was stretched on a tissue
stretching device and incubated in solutions in a stretched
configuration. For tubular tissues (e.g., ureters, arteries, veins,
and tubular SIS), the tissue was perfused with solutions using a
peristaltic pump and were soaked during incubation. The tissues
were incubated for 2 to 24 hours in 0.5% Triton X-100/0.5%
Deoxycholic acid with 5 mM EDTA in DPBS. The tissues were rinsed 3
times for 15-30 minutes each time in DPBS. The tissues were stored
at 4.degree. C. Within 48 hours of tissue storage, the tissues were
processed in supercritical CO.sub.2 for 20-120 minutes followed by
rapid depressurization (RDP)(decrease in pressure from 9.9 MPa to
0.69 MPa in 1 min 19 sec, corresponding to a depressurization of
2.7 MPa/10 sec).
TABLE-US-00002 TABLE 2 Triton X-100 Deoxycholic TX-100/Deoxy
Supercritical Material Conc. Acid Conc. incubation CO.sub.2 time
Porcine 0.5% 0.5% 24 hours 60 minutes ureters Bovine 0.5% 0.5% 24
hours 60 minutes pericardium Porcine 0.5% 0.5% 2 hours 60 minutes
mesothelium SIS 0.5% 0.5% 2 hours 60 minutes
[0100] The results showed that supercritical CO.sub.2 exposure
followed by rapid depressurization (SCCO.sub.2+RDP) did aid in the
removal of cell remnants and DNA while preserving growth factors in
the ECMs.
Example 8
[0101] The growth factor content of various ECM compositions was
analyzed using basic fibroblast growth factor (bFGF) as a
representative growth factor. bFGF was selected because it is a
prevalent growth factor in native ECM tissues. An enzyme-linked
immunosorbent assay (ELISA, R&D Systems, Minneapolis, Minn.)
was used to measure the bFGF content in the following samples: (1)
Unprocessed (Raw) SIS; (2) SIS after detergent soak (TX-deoxy)
only; (3) SIS after TX-deoxy and RDP (includes SCCO.sub.2); (4) SIS
after TX-deoxy, RDP, and PAA (SCCO.sub.2 with PAA for
sterilization); (5) SIS after TX-deoxy, and PAA; (6) SIS sterilized
by EtO (supplied by Cook Biotech, Inc.); and (7) non-sterile SIS
(supplied by Cook Biotech, Inc.).
[0102] In these studies, SIS was used to compare an ECM composition
processed with and without RDP to SIS provided by Cook Biotech,
Inc. Some of the processed SIS was also sterilized using the
described SCCO.sub.2+PAA method after decellularization. The
measured growth factor content of the respective ECM compositions
is shown in FIG. 7.
[0103] These results indicate that the rapid depressurization
process was more effective than other decellularization processes
at preserving the bFGF content and that the additional RDP
processing to remove residual DNA and cell fragments results in
only a small loss of bFGF. By comparison, the PAA sterilization
process appeared to remove almost all of the remaining bFGF, even
in the absence of RDP. Additionally, the rapid depressurization
process preserved more of the bFGF content in the native SIS than
the Cook decellularization methods. For purposes of these results,
when the bFGF content was reduced, it is assumed that all other
growth factor content was similarly reduced since the growth
factors are all bound to the ECM compositions in a similar
manner.
[0104] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0105] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
* * * * *