U.S. patent application number 15/843159 was filed with the patent office on 2018-08-16 for graft materials containing bioactive substances, and methods for their manufacture.
This patent application is currently assigned to Cook Biotech Incorporated. The applicant listed for this patent is Cook Biotech Incorporated. Invention is credited to David M.J. Ernst, Michael C. Hiles, Jason P. Hodde, Lal Ninan.
Application Number | 20180228939 15/843159 |
Document ID | / |
Family ID | 34272600 |
Filed Date | 2018-08-16 |
United States Patent
Application |
20180228939 |
Kind Code |
A1 |
Hiles; Michael C. ; et
al. |
August 16, 2018 |
GRAFT MATERIALS CONTAINING BIOACTIVE SUBSTANCES, AND METHODS FOR
THEIR MANUFACTURE
Abstract
Described are packaged, sterile medical graft products
containing controlled levels of a growth factor such as Fibroblast
Growth Factor-2 (FGF-2). Also described are methods of
manufacturing medical graft products wherein processing, including
sterilization, is controlled and monitored to provide medical graft
products having modulated, known levels of a extracellular matrix
factor, such as a growth factor, e.g. FGF-2. Preferred graft
materials are extracellular matrix materials isolated from human or
animal donors, particularly submucosa-containing extracellular
matrix materials. Further described are ECM compositions that are
or are useful for preparing gels, and related methods for
preparation and use.
Inventors: |
Hiles; Michael C.; (West
Lafayette, IN) ; Hodde; Jason P.; (West Lafayette,
IN) ; Ernst; David M.J.; (Dickinson, ND) ;
Ninan; Lal; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cook Biotech Incorporated |
West Lafayette |
IN |
US |
|
|
Assignee: |
Cook Biotech Incorporated
West Lafayette
IN
|
Family ID: |
34272600 |
Appl. No.: |
15/843159 |
Filed: |
December 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15333740 |
Oct 25, 2016 |
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15843159 |
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13225585 |
Sep 6, 2011 |
9504769 |
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15333740 |
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10569218 |
Dec 18, 2006 |
8021692 |
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PCT/US2004/027557 |
Aug 25, 2004 |
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13225585 |
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60497746 |
Aug 25, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/54 20130101;
A61P 43/00 20180101; A61L 2400/06 20130101; A61L 27/3633 20130101;
A61K 35/38 20130101; A61L 27/227 20130101; A61L 27/3629 20130101;
A61L 27/50 20130101; A61L 2300/414 20130101; A61L 27/3687 20130101;
A61K 38/1825 20130101; A61L 27/24 20130101 |
International
Class: |
A61L 27/54 20060101
A61L027/54; A61L 27/36 20060101 A61L027/36; A61L 27/50 20060101
A61L027/50; A61L 27/24 20060101 A61L027/24 |
Claims
1-10. (canceled)
11. A medical product, comprising a packaged, sterile extracellular
matrix material isolated from animal tissue and comprising
components native to said tissue including collagen, growth
factors, proteoglycans, and glycosaminoglycans, and having
extractable, bioactive Fibroblast Growth Factor-2 (FGF-2) at a
level of at least about 50 ng/g dry weight.
12. The medical product of claim 11, wherein the extracellular
matrix material comprises submucosa.
13. The medical product of claim 12, wherein the submucosa is small
intestinal submucosa.
14. The medical product of claim 12, wherein the submucosa is
porcine.
15. The medical product of claim 11, wherein the FGF-2 is present
at a level of at least about 60 ng/g dry weight.
16. The medical product of claim 11, wherein the FGF-2 is present
at a level of at least about 70 ng/g dry weight.
17. The medical product of claim 11, wherein the FGF-2 is present
at a level of at least about 80 ng/g dry weight.
18. The medical product of claim 11, wherein the FGF-2 is present
at a level of at least about 100 ng/g dry weight.
19. The medical product of claim 11, wherein the extracellular
matrix material has been sterilized with radiation.
20. The medical product of claim 19, wherein the material has been
sterilized with e-beam radiation.
21-105. (canceled)
106. A small intestinal submucosa medical product, comprising: a
sterile graft construct enclosed in a medical package; said graft
construct including at least one sheet of an extracellular matrix
material comprising small intestinal submucosa from a warm-blooded
vertebrate animal; said extracellular matrix material comprising
bioactive Fibroblast Growth Factor-2 (FGF-2) from a source tissue
for the extracellular matrix material; said extracellular matrix
material comprising bioactive Transforming Growth Factor-beta
(TGF-beta) from a source tissue for the extracellular matrix
material; said extracellular matrix material comprising bioactive
Vascular Endothelial Growth Factor (VEGF) from a source tissue for
the extracellular matrix material; and said extracellular matrix
material comprising glycosamnoglycans, glycoproteins and
proteoglycans from a source tissue for the extracellular matrix
material;
107. The medical product of claim 106, wherein the extracellular
matrix material is from a porcine animal.
108. The medical product of claim 106, wherein the graft construct
has been sterilized while enclosed in the medical package by
exposure to ethylene oxide gas, e-beam radiation or gas plasma.
109. The medical product of claim 108, wherein the graft construct
comprises a multilaminate construct including multiple sheets of an
extracellular matrix material comprising small intestinal submucosa
from a warm-blooded vertebrate animal.
110. The medical product of claim 109, wherein the graft construct
has a three-dimensional form.
111. The medical product of claim 110, wherein the
three-dimensional form is a generally tubular form.
112. A small intestinal submucosa medical product, comprising: a
sterile graft construct enclosed in a medical package; said graft
construct including at least one sheet of an extracellular matrix
material comprising small intestinal submucosa from a warm-blooded
vertebrate animal; said extracellular matrix material comprising
bioactive Fibroblast Growth Factor-2 (FGF-2) from a source tissue
for the extracellular matrix material; wherein the graft construct
has been sterilized while enclosed in the medical package by
exposure to ethylene oxide gas, with said exposure effective to
sterilize the graft construct while reducing the level of
extractable, bioactive FGF-2 in the extracellular matrix material
to less than 40% of a starting level of extractable, bioactive
FGF-2 present in the extracellular matrix material at the start of
said exposure.
113. The medical product of claim 112, wherein the graft construct
comprises a multilaminate construct including multiple sheets of an
extracellular matrix material comprising small intestinal submucosa
from a warm-blooded vertebrate animal.
114. The medical product of claim 112, wherein the graft construct
has a three-dimensional form.
115. The medical product of claim 115, wherein the
three-dimensional form is a generally tubular form.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/333,740, filed Oct. 25, 2016, pending,
which is a continuation of U.S. patent application Ser. No.
13/225,585, filed Sep. 6, 2011, now U.S. Pat. No. 9,504,769, which
is a continuation of U.S. patent application Ser. No. 10/569,218,
filed Dec. 18, 2006, now U.S. Pat. No. 8,021,692, which is the
National Stage of International Application No. PCT/US2004/027557,
filed Aug. 25, 2004, now abandoned, which claims the benefit of
U.S. Patent Application Ser. No. 60/497,746, filed Aug. 25, 2003,
now abandoned, each of which is hereby incorporated by reference in
its entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to materials useful
for tissue grafting, and in particular to such materials derived
from extracellular matrices and retaining both collagen and
substances such as growth factors that contribute to the beneficial
properties of the materials. In one aspect, the invention relates
to extracellular matrix tissue graft materials containing one or
more growth factors modulated to a predetermined level, and related
methods of manufacturing.
[0003] Extracellular matrix (ECM) materials, including those
derived from submucosa and other tissues, are known tissue graft
materials. See, e.g., U.S. Pat. Nos. 4,902,508, 4,956,178,
5,281,422, 5,372,821, 5,554,389, 6,099,567, and 6,206,931. Tissues
from various biological structures can be used for these purposes,
including for example small intestine, stomach, the urinary
bladder, skin, pericardium, dura mater, fascia, and the like. These
sources provide collagenous materials useful in a variety of
surgical procedures where tissue support and/or ingrowth are
desired.
[0004] Submucosa and other ECM materials have been shown to include
a variety of components other than collagen that that can
contribute to the bioactivity of the materials and to their value
in medical grafting and other uses. As examples, ECM materials can
include growth factors, cell adhesion proteins, and proteoglycans.
However, ECM materials are typically subjected to a battery of
manipulations in the manufacture of finished products containing
them. This presents challenges in obtaining finished products that
not only possess the necessary physical properties and appropriate
levels of biocompatibility and sterility, but also the desired
bioactivity. The present invention is addressed to these needs.
SUMMARY OF THE INVENTION
[0005] Accordingly, in one aspect, the present invention provides a
method for manufacturing a tissue graft material such as a
collagenous extracellular matrix containing at least one
extractable, bioactive growth factor or other non-collagenous
protein material, particularly Fibroblast Growth Factor-2 (FGF-2),
at a predetermined amount. The method includes the steps of
providing a non-sterile extracellular matrix material; fashioning a
plurality of graft products from the extracellular matrix material;
packaging the products; subjecting the packaged products to a
sterilization procedure that affects the level of extractable
bioactive growth factor (FGF-2) or other non-collagenous protein
material in the products; and, taking and testing sample products
of the sterilized packaged products to determine a level of a
growth factor (FGF-2) in the sample products, wherein said
determined level is representative of an approximate level of said
growth factor in other ones of said products from the lot from
which the sample product was taken.
[0006] In another aspect, the present invention provides a medical
product that comprises a packaged, sterile animal-derived
extracellular matrix material comprising FGF-2 at a level of at
least about 50 nanograms per gram dry weight. Particularly
preferred materials are lyophilized and/or include submucosa.
[0007] Another aspect of the invention provides a packaged, sterile
extracellular matrix material isolated from animal tissue and
including components native to the tissue, the matrix material
including collagen, growth factors, proteoglycans,
glycosaminoglycans, and having extractable, bioactive FGF-2 at a
level of at least about 50 nanograms per gram dry weight.
[0008] Another aspect of the invention provides a method for
manufacturing a sterile, extracellular matrix material. The method
includes isolating an extracellular matrix material from animal
tissue, the isolated extracellular matrix material including
extractable FGF-2 at a first level; and, sterilizing the isolated
extracellular matrix material under conditions to retain the
extractable, bioactive FGF-2 in at least 10% of the first
level.
[0009] Another aspect of the invention provides a method for
manufacturing medical products. The method includes providing
extracellular matrix material in non-sterile condition and isolated
from animal tissue, the extracellular matrix material comprising
extractable, bioactive FGF-2; packaging and sterilizing the
extracellular matrix material to provide product lots each
containing multiple, packaged extracellular matrix material
products; taking sample products from the product lots; and testing
the sample products to determine whether they include extractable,
bioactive FGF-2 at a level above a predetermined level, e.g. above
about 50 nanograms per gram dry weight.
[0010] Another aspect of the invention provides a medical product
adapted for treating wounds, the product including an extracellular
matrix material isolated from animal tissue, the material including
bioactive components useful to treat wounds including but not
limited to FGF-2. The FGF-2 is present in the extracellular matrix
material at a level of at least about 50 nanograms per gram dry
weight.
[0011] Another aspect of the invention relates to a medical product
comprising a dry collagenous powder comprising extracellular matrix
material, wherein the dry collagenous powder is effective to gel
upon rehydration with an aqueous medium and comprises FGF-2 at a
level of at least about 50 ng/g dry weight.
[0012] In another aspect, the invention relates to a medical
product comprising a fluid composition comprising solubilized or
suspended collagenous extracellular matrix material, wherein the
fluid composition comprises FGF-2 at a level of about 0.1 ng/ml to
about 100 ng/ml.
[0013] In another embodiment, the invention provides a method for
disinfecting an aqueous extracellular matrix hydrolysate
composition. The aqueous extracellular matrix hydrolysate
composition is contacted with an oxidizing disinfectant for a
period of time and under conditions sufficient to disinfect the
aqueous extracellular matrix hydrolysate composition.
[0014] Another aspect of the invention relates to a method for
preparing a disinfected, extracellular matrix hydrolysate
composition. This method comprises forming an aqueous extracellular
matrix hydrolysate. A first dialysis step is conducted and includes
dialyzing the aqueous extracellular matrix hydrolysate against an
aqueous medium containing an oxidizing disinfectant so as to
contact and disinfect the extracellular matrix hydrolysate with the
oxidizing disinfectant and thereby form a disinfected extracellular
matrix hydrolysate. A second dialysis step includes dialyzing the
disinfected extracellular matrix hydrolysate under conditions to
remove the oxidizing disinfectant.
[0015] In another embodiment, the invention provides an
extracellular matrix hydrolysate product having extracellular
matrix components disinfected by contact of an aqueous medium
containing the extracellular matrix hydrolysate with an oxidizing
disinfectant. The extracellular matrix hydrolysate product can take
on a variety of forms, including a dry powdery material, a
non-gelled aqueous composition, a gel, or a sponge.
[0016] Still another embodiment of the invention provides an
extracellular matrix graft material that includes an extracellular
matrix hydrolysate combined with extracellular matrix particles. In
a preferred form, the graft material includes an aqueous medium
having said extracellular matrix hydrolysate in a dissolved state
with the extracellular matrix particles suspended therein,
desirably wherein the medium exhibits gel-forming capacity.
[0017] Another embodiment of the invention provides an
extracellular matrix graft material that includes a sterile,
injectable fluid extracellular matrix composition including an
aqueous medium containing an extracellular matrix hydrolysate. The
extracellular matrix hydrolysate is present in the composition at a
level of at least about 20 mg/ml, for example in the range of about
20 mg/ml to about 200 mg/ml.
[0018] Additional aspects as well as features and advantages of the
invention will be apparent to those of ordinary skill in the art
from the descriptions herein.
DETAILED DESCRIPTION
[0019] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to certain
embodiments and specific language will be used to describe the
same. It will nevertheless be understood that no limitation of the
scope of the invention is thereby intended, such alterations and
further modifications in the illustrated device, and such further
applications of the principles of the invention as described herein
being contemplated as would normally occur to one skilled in the
art to which the invention relates.
[0020] As disclosed above, in one aspect, the present invention
provides packaged, sterile medical products including tissue grafts
materials containing one or more growth factors, and methods for
manufacturing the same. As for the tissue graft material used, it
will desirably be a naturally-derived material such as an
extracellular matrix (ECM) material. Preferred are
naturally-derived collagenous ECMs isolated from suitable animal or
human tissue sources. Suitable extracellular matrix materials
include, for instance, submucosa (including for example small
intestinal submucosa, stomach submucosa, urinary bladder submucosa,
or uterine submucosa, each of these isolated from juvenile or adult
animals), renal capsule membrane, amnion, dura mater, pericardium,
serosa, peritoneum or basement membrane materials, including liver
basement membrane or epithelial basement membrane materials. These
materials may be isolated and used as intact natural sheet forms,
or reconstituted collagen layers including collagen derived from
these materials and/or other collagenous materials may be used. For
additional information as to submucosa materials useful in the
present invention, and their isolation and treatment, reference can
be made to U.S. Pat. Nos. 4,902,508, 5,554,389, 5,993,844,
6,206,931, and 6,099,567. Renal capsule membrane can also be
obtained from warm-blooded vertebrates, as described more
particularly in International Patent Application serial No.
PCT/US02/20499 filed Jun. 28, 2002, published Jan. 9, 2003 as
WO03002165.
[0021] Preferred ECM base materials contain residual bioactive
proteins or other ECM components derived from the tissue source of
the materials. For example, they may contain Fibroblast Growth
Factor-2 (basic FGF), Transforming Growth Factor-beta (TGF-beta)
and vascular endothelial growth factor (VEGF). It is also expected
that ECM base materials of the invention may contain additional
bioactive components including, for example, one or more of
glycosaminoglycans, glycoproteins, proteoglycans, and/or growth
factors.
[0022] It has been discovered that the sterilization conditions
utilized in the manufacture of tissue graft materials can
significantly impact the level of one or more of such bioactive
components or growth factors, including for example FGF-2.
Accordingly, in accordance with the invention, sterilization
protocols can be selected and controlled to modulate the level of
growth factors, for example by either intentionally reducing growth
factor levels to a predetermined level or below, or to retain at
least a given percentage or level of one or more growth factors,
particularly FGF-2, in the material. In certain embodiments of the
invention, the ECM or other graft material is processed to
finished, packaged, sterile products containing FGF-2 at a level of
at least 50 ng/g dry weight, or even at least about 60, at least
about 70, at least about 80, or at least about 100 ng/g dry weight.
In other embodiments of the invention, an ECM material will have a
first level of a bioactive component, such as FGF-2 or another
growth factor, after isolation from the animal or human donor
source tissue and rinsing with a rinse agent such as water. The ECM
material will thereafter be processed under controlled conditions,
including sterilization, to provide packaged, sterile medical
products containing at least about 10% of said first level of the
FGF-2 or other bioactive component, or even at least 15%, 20%, 30%
or even 50% or more of said first level.
[0023] Illustratively, it has been found that sterilization
protocols including ethylene oxide (EO) sterilization, electron
beam (E-beam) radiation and gas plasma sterilization (e.g.
Sterrad.RTM.) can significantly reduce levels of extractable,
bioactive FGF-2. At the same time, these sterilization techniques
have significantly lower or essentially no impact on levels of
extractable, bioactive TGF-beta. Advantageously, the modulation of
growth factors imparted by the sterilization technique can be used
to affect and optimize levels of given growth factors, their
ratios, etc., to prepare a graft material better suited for a
particular medical indication wherein the retained growth factor or
growth factors are beneficial to the indication, and/or wherein
eliminated growth factor or growth factors are deleterious to the
medical indication.
[0024] For example, FGF-2 is known to stimulate angiogenesis,
neurite growth, plasminogen activator (PA) secretion, and matrix
metalloproteinase 1 (MMP-1) production. Correspondingly, levels of
FGF-2 can be retained and optimized for use in the graft material
in wound healing (angiogenesis), treatment of nervous tissue
(neurite outgrowth) including peripheral nervous tissue and central
nervous tissue, modulating adhesion formation (by stimulating PA),
and facilitating collagen turnover and degradation (by stimulating
MMP-1 production). Thus, FGF-2 levels can be retained in the
material as high as possible by selecting and optimizing the
sterilization protocol. For instance, it has been found that
non-sterile isolated submucosa layers (and in particular isolated
from small intestine), contain relatively high levels of
extractable, bioactive FGF-2. For example, submucosa tissue
isolated from small intestine and minimally treated, e.g. only by
rinsing, may be recovered so as to contain in excess of about 100
nanograms per gram of FGF-2 dry weight and potentially even higher
levels such as above about 200 or about 400 nanograms per gram. In
manufacturing, it may be beneficial to retain as much of this FGF-2
in the material as possible. Thus, intermediate steps between the
isolation of the original submucosa material and the finished,
packaged medical article, can be selected and controlled so as to
maintain as much active FGF-2 in the material as possible.
[0025] As one example, an isolated, small intestinal submucosa
material disinfected as described in U.S. Pat. No. 6,206,931 with
peracetic acid may contain from about 70 to about 200 nanograms per
gram (dry weight) of FGF-2. It has been found that sterilization
treatments using ethylene oxide, E-beam, and gas plasma
sterilization techniques significantly reduce the levels of FGF-2
in the disinfected material. Among these, E-beam sterilization had
the smallest impact on FGF-2 levels, with E-beam sterilized
submucosa having FGF-2 levels ranging from about 75 nanograms per
gram dry weight to about 150 nanograms per gram dry weight, and
generally retaining greater than about 50% of the FGF-2 level of
the disinfected submucosa material. Gas plasma sterilized material
had an FGF-2 level ranging from about 60 nanograms per gram dry
weight to about 110 nanograms per gram dry weight, and retaining at
least 40% of the FGF-2 level of the disinfected submucosa material.
Thus, in embodiments of the invention, materials sterilized using
E-beam or gas plasma techniques are used in products configured for
and methods for treating patients where relatively high FGF-2
levels are beneficial, for example wound healing, treatment of
tissue of the nervous system, modulating adhesions, or facilitating
collagen turnover and degradation.
[0026] On the other hand, ethylene oxide sterilization at both low
temperature and high temperature conditions had a more significant
impact in reducing the FGF-2 levels, with products typically having
from about 10 to about 40 nanograms per gram of FGF-2 dry weight,
and retaining less than about 40% of the FGF-2 level of the
disinfected submucosa material (e.g. about 10% to about 40%). In
this ethylene oxide work, the high temperature conditions tended to
do have a slightly greater effect in reducing the FGF-2 levels than
the low temperature conditions. Accordingly, in the ethylene oxide
and potentially other sterilization techniques, the temperature may
be increased or decreased to provide a respective higher or lower
level of reduction of FGF-2 and/or other growth factors or
non-collagenous ECM proteins. Similarly, the total dose of
sterilant chemical or energy can be increased or decreased to
provide a respective higher or lower level of reduction of FGF-2
and/or other growth factors or non-collagenous ECM proteins.
Increased doses of sterilant can be achieved, for instance, through
a longer, single exposure of the graft material to the sterilant,
or through multiple, discreet exposures of the graft material to
the sterilant.
[0027] In accordance with the invention, in addition to controlling
the sterilization protocol, a number of other manufacturing
techniques can be undertaken to provide a packaged, sterilized
graft product with a controlled level of one or more growth
factors, including for example FGF-2. As a first measure, where it
is desired to retain as high as possible a level of FGF-2, the
animal-derived collagenous ECM can be processed and preserved from
the time of harvest to the time at which FGF-2 or other growth
factor is protected against further significant degradation. For
these purposes, the harvested tissue from which the ECM material is
to be isolated may be placed soon or immediately after harvest in a
stabilizing solution that prevents degradation of the product
including for example, osmotic, hypoxic, autolytic, and/or
proteolytic degradation. This solution can also protect against
bacterial contamination. To achieve these effects, the stabilizing
material may be a buffered solution of anti-oxidants, antibiotics,
protease inhibitors, oncotic agents, or other stabilizing
agents.
[0028] Illustratively, enzymes (e.g. superoxide dismutase and
catalase) may be used to neutralize the superoxide anion and
hydrogen peroxide or compounds that can directly react with and
neutralize other free-radical species. Antioxidants may be added
and include tertiary butylhydroquinone (BHT), alpha tocopherol,
mannitol, hydroxyurea, glutathione, ascorbate,
ethylenediaminetetraacetic acid (EDTA) and the amino acids
histidine, proline and cysteine. In addition to antioxidants, the
stabilizing solution may contain agents to inhibit hypoxic
alteration to normal biochemical pathways, for example, allopurinol
to inhibit xanthine dehydrogenase, lipoxigenase inhibitors, calcium
channel blocking drugs, calcium binding agents, iron binding
agents, metabolic intermediaries and substrates of adenosine
triphosphate (ATP) generation.
[0029] The stabilizing solution may also contain one or more
antibiotics, antifungal agents, protease inhibitors, proteoglycans,
and an appropriate buffer. Antibiotics can be used to inhibit or
prevent bacterial growth and subsequent tissue infection.
Antibiotics may be selected from the group of penicillin,
streptomycin, gentamicin, kanamycin, neomycin, bacitracin, and
vancomycin. Additionally, anti-fungal agents may be employed,
including amphotericin-B, nystatin and polymyxin.
[0030] Protease inhibitors may be included in the stabilizing
solution to inhibit endogenous proteolytic enzymes which, when
released, can cause irreversible degradation of the ECM, as well as
the release of chemoattractant factors. These chemoattractants
solicit the involvement of polymorphonuclear leukocytes,
macrophages and other natural killer cells which generate a
nonspecific immune response that can further damage the ECM.
Protease inhibitors can be selected from the group consisting of
N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF),
ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis
(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), leupeptin,
ammonium chloride, elevated pH and apoprotinin.
[0031] Glycosaminoglycans may be included in the stabilizing
solution to provide a colloid osmotic balance between the solution
and the tissue, thereby preventing the diffusion of endogenous
glycosaminoglycans from the tissue to the solution. Endogenous
glycosaminoglycans serve a variety of functions in collagen-based
connective tissue physiology. They may be involved in the
regulation of cell growth and differentiation (e.g. heparin sulfate
and smooth muscle cells) or, alternatively, they are important in
preventing pathological calcification (as with heart valves).
Glycosaminoglycans are also involved in the complex regulation of
collagen and elastin synthesis and remodeling, which is fundamental
to connective tissue function. Glycosaminoglycans are selected from
the group of chondroitin sulfate, heparin sulfate, and dermatan
sulfate and hyaluronan. Non-glycosaminoglycan osmotic agents which
may also be included are polymers such as dextran and polyvinyl
pyrolodone (PVP) and amino acids such as glycine and proline.
[0032] The stabilizing solution can also contain an appropriate
buffer. The nature of the buffer is important in several aspects of
the processing technique. Crystalloid, low osmotic strength buffers
have been associated with damage occurring during saphenous vein
procurement and with corneal storage. Optimum pH and buffering
capacity against the products of hypoxia damage (described below),
is essential. In this context the organic and bicarbonate buffers
have distinct advantages. (In red cell storage, acetate-citrate
buffers with glycine and glucose have been shown to be effective in
prolonging shelf-life and maintaining cellular integrity.) The
inventors prefer to use an organic buffer selected from the group
consisting of 2-(N-morpholino)ethanesulfonic acid (MES),
3-(N-morpholine)propanesulfonic acid (MOPS) and
N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES).
Alternatively, a low salt or physiological buffer, including
phosphate, bicarbonate and acetate-citrate, may be more appropriate
in certain applications.
[0033] In another aspect, components of the stabilizing solution
address one or more of the events that occur during the harvesting
of tissues, such as spasm, hypoxia, hypoxia reperfusion, lysosomal
enzyme release, platelet adhesion, sterility and buffering
conditions. Involuntary contraction of the smooth muscles can
result from mechanical stretching or distension, as well as from
the chemical action of certain endothelial cell derived contraction
factors, typically released under hypoxic (low oxygen) conditions.
This involuntary contraction may result in damage to the adjacent
ECM. For this reason, the stabilizing solution can include one or
more smooth muscle relaxants, selected from the group of calcitonin
gene related peptide (CGRP), papaverine, sodium nitroprusside
(NaNP), H7 (a protein Kinase C inhibitor) calcium channel blockers,
calcium chelators, isoproterenol, phentolamine, pinacidil,
isobutylmethylxanthine (IBMX), nifedepine and flurazine. The
harvested tissue can be immediately placed into this stabilizing
solution and is maintained at 4.degree. C. during transportation
and any storage prior to further processing.
[0034] The tissue graft material of the invention can be provided
in any suitable form, including substantially two-dimensional sheet
form (optionally meshed sheet), or a three-dimensional form such as
a tube, valve leaflet, or the like. The tissue graft material may
contain a single layer of isolated ECM material, or may be a
multilaminate construct sized the same as its component layers
(e.g. containing directly overlapped layers) or larger than its
component layers (e.g. containing partially overlapped layers),
see, e.g., U.S. Pat. Nos. 5,885,619 and 5,711,969.
[0035] In another embodiment, the invention provides a medical
product that includes a dry collagenous powder useful for example
to treat wounds or to otherwise induce tissue growth at a desired
implant location, and including an ECM material. The powder is
desirably effective to gel upon rehydration with an aqueous medium
and includes FGF-2 at a level of at least about 50 nanograms per
gram dry weight. Illustratively, the powder can include a
particulate of ECM material prepared by drying a fluidized material
prepared as described in U.S. Pat. Nos. 5,516,533 and 5,275,826.
This resulting powder can be used alone or in combination with
other powder materials to support gelling of the overall powder
upon rehydration with an aqueous medium such as a buffered saline
solution. In this regard, in addition to the particulate ECM
material, the powder composition may also include powdered,
purified collagen, gelatin, or the like, to assist in gelling the
product upon rehydration.
[0036] In one embodiment, the preparation of the powder will be
conducted to include FGF-2 at a level of at least about 50
nanograms per gram dry weight, more preferably at least about 60,
70, 80, or 100 nanograms per gram dry weight. Resultant fluid
compositions containing solubilized or suspended collagenous ECM
materials will desirably be prepared to contain FGF-2 at a level of
about 0.1 nanograms per milliliter or greater, e.g. typically in
the range of about 0.1 nanograms per ml to about 100 nanograms per
ml. This fluid composition is desirably gelable, for example upon
incubation for a time after rehydration, which may be hastened by
bringing the fluid composition to a relatively neutral pH and/or to
body temperature from room temperature. In other embodiments, the
fluidized medical product may contain FGF-2 at a level of about 1
to about 15 nanograms per ml, or may contain FGF-2 at a level of
about 10 to about 30 nanograms per ml.
[0037] As disclosed above, certain embodiments of the invention
provide packaged, sterile medical products. Known packaging
techniques and materials can be used in the manufacture of such
products, with the packaging being selected to suit the final
sterilization technique being employed, e.g. ethylene oxide gas,
electron-beam, or gas plasma techniques. In addition, the packaging
may contain or otherwise bear indicia relating to the use of the
enclosed graft material for a particular medical indication, e.g.
wound care, and/or may contain or otherwise bear indicia as to one
or more growth factors (e.g. FGF-2) for which the product
manufacture has been controlled to modulate its level, e.g. to
reflect a minimum level of such growth factor, a maximum level of
such growth factor, or a range of such growth factor contained in
the enclosed tissue graft product.
[0038] In other embodiments, the present invention provides ECM gel
compositions and methods and materials for their preparation, which
can optionally also be used in conjunction with the techniques
described above for modulating the level of one or more bioactive
substances in the product, including for example growth factors
such as FGF-2. The gel compositions of the invention can be
prepared from an isolated ECM material, for example one of those
listed above. The ECM material is used to prepare a solubilized
mixture including components of the material. This can be achieved
by digestion of the ECM material in an acidic or basic medium
and/or by contact with an appropriate enzyme or combination of
enzymes.
[0039] Typically, the ECM material is reduced to particulate form
to aid in the digestion step. This can be achieved by tearing,
cutting, grinding or shearing the isolated ECM material.
Illustratively, shearing may be conducted in a fluid medium, and
grinding may be conducted with the material in a frozen state. For
example, the material can be contacted with liquid nitrogen to
freeze it for purposes of facilitating grinding into powder form.
Such techniques can involve freezing and pulverizing submucosa
under liquid nitrogen in an industrial blender.
[0040] Any suitable enzyme may be used for an enzymatic digestion
step. Such enzymes include for example serine proteases, aspartyl
proteases, and matrix metalloproteases. The concentration of the
enzyme can be adjusted based on the specific enzyme used, the
amount of submucosa to be digested, the duration of the digestion,
the temperature of the reaction, and the desired properties of the
final product. In one embodiment about 0.1% to about 0.2% of enzyme
(pepsin, for example) is used and the digestion is conducted under
cooled conditions for a period of time sufficient to substantially
digest the ECM material. The digestion can be conducted at any
suitable temperature, with temperatures ranging from 4-37.degree.
C. being preferred. Likewise, any suitable duration of digestion
can be used, such durations typically falling in the range of about
2-180 hours. The ratio of the concentration of ECM material
(hydrated) to total enzyme usually ranges from about 25 to about
125 and more typically the ratio is about 50, and the digestion is
conducted at 4.degree. C. for 24-72 hours. When an enzyme is used
to aid in the digestion, the digestion will be performed at a pH at
which the enzyme is active and more advantageously at a pH at which
the enzyme is optimally active. Illustratively, pepsin exhibits
optimal activity at pH's in the range of about 2-4.
[0041] The enzymes or other disruptive agents used to solubilize
the ECM material can be removed or inactivated before or during the
gelling process so as not to compromise gel formation or subsequent
gel stability. Also, any disruptive agent, particularly enzymes,
that remain present and active during storage of the tissue will
potentially change the composition and potentially the gelling
characteristics of the solution. Enzymes, such as pepsin, can be
inactivated with protease inhibitors, a shift to neutral pH, a drop
in temperature below 0.degree. C., heat inactivation or through the
removal of the enzyme by fractionation. A combination of these
methods can be utilized to stop digestion of the ECM material at a
predetermined endpoint, for example the ECM material can be
immediately frozen and later fractionated to limit digestion.
[0042] The ECM material is enzymatically digested for a sufficient
time to produce a hydrolysate of ECM components. The ECM can be
treated with one enzyme or with a mixture of enzymes to hydrolyze
the structural components of the material and prepare a hydrolysate
having multiple hydrolyzed components of reduced molecular weight.
The length of digestion time is varied depending on the
application, and the digestion can be extended to completely
solubilize the ECM material. In some modes of operation, the ECM
material will be treated sufficiently to partially solubilize the
material to produce a digest composition comprising hydrolyzed ECM
components and nonhydrolyzed ECM components. The digest composition
can then optionally be further processed to remove at least some of
the nonhydrolyzed components. For example, the nonhydrolyzed
components can be separated from the hydrolyzed portions by
centrifugation, filtration, or other separation techniques known in
the art.
[0043] Preferred gel compositions of the present invention are
prepared from enzymatically digested vertebrate ECM material that
has been fractionated under acidic conditions, for example
including pH ranging from about 2 to less than 7, especially to
remove low molecular weight components. Typically, the ECM
hydrolysate is fractionated by dialysis against a solution or other
aqueous medium having an acidic pH, e.g. a pH ranging from about 2
to about 5, more desirably greater than 3 and less than 7. In
addition to fractionating the hydrolysate under acidic conditions,
the ECM hydrolysate is typically fractionated under conditions of
low ionic strength with minimal concentrations of salts such as
those usually found in standard buffers such as PBS (i.e. NaCl,
KCl, Na.sub.2HPO.sub.4, or KH.sub.2PO.sub.4) that can pass through
the dialysis membrane and into the hydrolysate. Such fractionation
conditions work to reduce the ionic strength of the ECM hydrolysate
and thereby provide enhanced gel forming characteristics.
[0044] The hydrolysate solution produced by enzymatic digestion of
the ECM material has a characteristic ratio of protein to
carbohydrate. The ratio of protein to carbohydrate in the
hydrolysate is determined by the enzyme utilized in the digestion
step and by the duration of the digestion. The ratio may be similar
to or may be substantially different from the protein to
carbohydrate ratio of the undigested ECM tissue. For example,
digestion of vertebrate ECM material with a protease such as
pepsin, followed by dialysis, will form a fractionated ECM
hydrolysate having a lower protein to carbohydrate ratio relative
to the original ECM material.
[0045] In accordance with certain embodiments of the invention,
shape retaining gel forms of ECM are prepared from ECM material
that has been enzymatically digested and fractionated under acidic
conditions to form an ECM hydrolysate that has a protein to
carbohydrate ratio different than that of the original ECM
material. Such fractionation can be achieved entirely or at least
in part by dialysis. The molecular weight cut off of the ECM
components to be included in the gel material is selected based on
the desired properties of the gel. Typically the molecular weight
cutoff of the dialysis membrane (the molecular weight above which
the membrane will prevent passage of molecules) is within in the
range of about 2000 to about 10000 Dalton, and more preferably from
about 3500 to about 5000 Dalton.
[0046] In one embodiment of the invention, apart from the potential
removal of undigested ECM components after the digestion step and
any controlled fractionation to remove low molecular weight
components as discussed above, the ECM hydrolysate is processed so
as to avoid any substantial further physical separation of the ECM
components. For example, when a more concentrated ECM hydrolysate
material is desired, this can be accomplished by removing water
from the system (e.g. by evaporation or lyophilization) as opposed
to using conventional "salting out"/centrifugation techniques that
would demonstrate significant selectivity in precipitating and
isolating collagen, leaving behind amounts of other desired ECM
components. Thus, in certain embodiments of the invention,
solubilized ECM components of the ECM hydrolysate remain
substantially unfractionated, or remain substantially
unfractionated above a predetermined molecular weight cutoff such
as that used in the dialysis membrane, e.g. above a given value in
the range of about 2000 to 10000 Dalton, more preferably about 3500
to about 5000 Dalton.
[0047] Vertebrate ECM material can be stored frozen (e.g. at about
-20 to about -80.degree. C.) in either its solid, comminuted or
enzymatically digested forms prior to formation of the gel
compositions of the present invention, or the material can be
stored after being hydrolyzed and fractionated. The ECM material
can be stored in solvents that maintain the collagen in its native
form and solubility. For example, one suitable storage solvent is
0.01 M acetic acid, however other acids can be substituted, such as
0.01 N HCl. In accordance with one embodiment the fractionated ECM
hydrolysate is dried (by lyophilization, for example) and stored in
a dehydrated/lyophilized state. The dried form can be rehydrated
and gelled to form a gel of the present invention.
[0048] In accordance with one embodiment, the fractionated ECM
hydrolysate will exhibit the capacity to gel upon adjusting the pH
of a relatively more acidic aqueous medium containing it to about 5
to about 9, more preferably about 6.6 to about 8.0, and typically
about 7.2 to about 7.8, thus inducing fibrillogenesis and matrix
gel assembly. In one embodiment, the pH of the fractionated
hydrolysate is adjusted by the addition of a buffer that does not
leave a toxic residue, and has a physiological ion concentration
and the capacity to hold physiological pH. Examples of suitable
buffers include PBS, HEPES, and DMEM. In one embodiment the pH of
the fractionated ECM hydrolysate is raised by the addition of a
buffered NaOH solution to 6.6 to 8.0, more preferably 7.2 to 7.8.
Any suitable concentration of NaOH solution can be used for these
purposes, for example including about 0.05 M to about 0.5 M NaOH.
In accordance with one embodiment, the ECM hydrolysate is mixed
with a buffer and sufficient 0.25 N NaOH is added to the mixture to
achieve the desired pH. If desired at this point, the resultant
mixture can be aliquoted into appropriate forms or into designated
cultureware and incubated at 37.degree. C. for 0.5 to 1.5 hours to
form an ECM gel.
[0049] The ionic strength of the ECM hydrolysate is believed to be
important in maintaining the fibers of collagen in a state that
allows for fibrillogenesis and matrix gel assembly upon
neutralization of the hydrolysate. Accordingly, if needed, the salt
concentration of the ECM hydrolysate material can be reduced prior
to neutralization of the hydrolysate. The neutralized hydrolysate
can be caused to gel at any suitable temperature, e.g. ranging from
about 4.degree. C. to about 40.degree. C. The temperature will
typically affect the gelling times, which may range from 5 to 120
minutes at the higher gellation temperatures and 1 to 8 hours at
the lower gellation temperatures. Typically, the hydrolysate will
be gelled at elevated temperatures to hasten the gelling process,
for example at 37.degree. C. In this regard, preferred neutralized
ECM hydrolysates will be effective to gel in less than about ninety
minutes at 37.degree. C., for example approximately thirty to
ninety minutes at 37.degree. C. Alternatively, the gel can be
stored at 4.degree. C., and under these conditions the setting of
the gel will be delayed, e.g. for about 3-8 hours.
[0050] Additional components can be added to the hydrolysate
composition before, during or after forming the gel. For example,
proteins carbohydrates, growth factors, therapeutics, bioactive
agents, nucleic acids, cells or pharmaceuticals can be added. In
certain embodiments, such materials are added prior to formation of
the gel. This may be accomplished for example by forming a dry
mixture of a powdered ECM hydrolysate with the additional
component(s), and then reconstituting and gelling the mixture, or
by incorporating the additional component(s) into an aqueous,
ungelled composition of the ECM hydrolysate before, during (e.g.
with) or after addition of the neutralization agent. In other
embodiments, the additional component(s) are added to the formed
ECM gel, e.g. by infusing or mixing the component(s) into the gel
and/or coating them onto the gel.
[0051] In one embodiment of the invention, a particulate ECM
material will be added to the hydrolysate composition, which will
then be incorporated in the formed gel. Such particulate ECM
materials can be prepared by cutting, tearing, grinding or
otherwise comminuting an ECM starting material. For example, a
particulate ECM material having an average particle size of about
50 microns to about 500 microns may be included in the hydrolysate,
more preferably about 100 microns to about 400 microns. The ECM
particulate can be added in any suitable amount relative to the
hydrolysate, with preferred ECM particulate to ECM hydrolysate
weight ratios (based on dry solids) being about 0.1:1 to about
200:1, more preferably in the range of 1:1 to about 100:1. The
inclusion of such ECM particulates in the ultimate gel can serve to
provide additional material that can function to provide
bioactivity to the gel (e.g. itself including FGF-2 and/or other
growth factors or bioactive substances as discussed herein) and/or
serve as scaffolding material for tissue ingrowth.
[0052] In certain embodiments, an ECM hydrolysate material to be
used in tissue augmentation, e.g. in functional or cosmetic
purposes, will incorporate an ECM particulate material. In these
embodiments, the ECM particulate material can be included at a size
and in an amount that effectively retains an injectable character
to the hydrolysate composition, for example by injection through a
needle having a size in the range of 18 to 31 gauge (internal
diameters of 0.047 inches to about 0.004 inches). In this fashion,
non-invasive procedures for tissue augmentation will be provided,
which in preferred cases will involve the injection of an ungelled
ECM hydrolysate containing suspended ECM particles at a relatively
lower (e.g. room) temperature, which will be promoted to form a
gelled composition when injected into a patient and thereby brought
to physiologic temperature (about 37.degree. C.).
[0053] In other aspects of the invention, it has been discovered
that processing techniques that involve contacting the ECM material
with a disinfecting oxidizing agent compound can significantly
affect not only the concentration of bioactive substances but also
the gelling quality of the collagen molecules. In particular, it
has been found that contacting an ECM material with an oxidizing
agent such as peracetic acid prior to digestion to form the ECM
hydrolysate can disrupt or impair the ability of ECM hydrolysate to
form a gel. On the other hand, contacting an aqueous medium
including ECM hydrolysate components with an oxidizing disinfectant
such as a peroxy compound provides an improved ability to recover a
disinfected ECM hydrolysate that exhibits the capacity to form
beneficial gels. In accordance with one embodiment of the
invention, an aqueous medium containing ECM hydrolysate components
is disinfected by providing a peroxy disinfectant in the aqueous
medium. This is advantageously achieved using dialysis to deliver
the peroxy disinfectant into and/or to remove the peroxy
disinfectant from the aqueous medium containing the hydrolysate. In
one preferred embodiment, the aqueous medium containing the ECM
hydrolysate is dialyzed against an aqueous medium containing the
peroxy disinfectant to deliver the disinfectant into contact with
the ECM hydrolysate, and then is dialyzed against an appropriate
aqueous medium (e.g. an acidic aqueous medium) to at least
substantially remove the peroxy disinfectant from the ECM
hydrolysate. During this dialysis step, the peroxy compound passes
through the dialysis membrane and into the ECM hydrolysate, and
contacts ECM components for a sufficient period of time to
disinfect the ECM components of the hydrolysate. In this regard,
typical contact times will range from about 0.5 hours to about 8
hours and more typically about 1 hour to about 4 hours. The period
of contact will be sufficient to substantially disinfect the
digest, including the removal of endotoxins and inactivation of
virus material present. The removal of the peroxy disinfectant by
dialysis may likewise be conducted over any suitable period of
time, for example having a duration of about 4 to about 180 hours,
more typically about 24 to about 96 hours. In general, the
disinfection step will desirably result in a disinfected ECM
hydrolysate composition having sufficiently low levels of
endotoxins, viral burdens, and other contaminant materials to
render it suitable for medical use. Endotoxin levels below about 2
endotoxin units (EUs) per gram (dry weight) are preferred, more
preferably below about 1 EU per gram, as are virus levels below 100
plaque forming units per gram (dry weight), more preferably below 1
plaque forming unit per gram.
[0054] In one embodiment, the aqueous ECM hydrolysate composition
is a substantially homogeneous solution during the dialysis step
for delivering the oxidizing disinfectant to the hydrolysate
composition and/or during the dialysis step for removing the
oxidizing disinfectant from the hydrolysate composition.
Alternatively, the aqueous hydrolysate composition can include
suspended ECM hydrolysate particles, optionally in combination with
some dissolved ECM hydrolysate components, during either or both of
the oxidizing disinfectant delivery and removal steps. Dialysis
processes in which at least some of the ECM hydrolysate components
are dissolved during the disinfectant delivery and/or removal steps
are preferred and those in which substantially all of the ECM
hydrolysate components are dissolved are more preferred.
[0055] The disinfection step can be conducted at any suitable
temperature, and will typically be conducted between 0.degree. C.
and 37.degree. C., more typically between about 4.degree. C. and
about 15.degree. C. During this step, the concentration of the ECM
hydrolysate solids in the aqueous medium is typically in the range
of about 2 mg/ml to about 200 mg/ml, and may vary somewhat through
the course of the dialysis due to the migration of water through
the membrane. In certain embodiments of the invention, a relatively
unconcentrated digest is used, having a starting ECM solids level
of about 5 mg/ml to about 15 mg/ml. In other embodiments of the
invention, a relatively concentrated ECM hydrolysate is used at the
start of the disinfection step, for example having a concentration
of at least about 20 mg/ml and up to about 200 mg/ml, more
preferably at least about 100 mg/ml and up to about 200 mg/ml. It
has been found that the use of concentrated ECM hydrolysates during
this disinfection processing results in an ultimate gel composition
having higher gel strength than that obtained using similar
processing with a lower concentration ECM hydrolysate. Accordingly,
processes which involve the removal of amounts of water from the
ECM hydrolysate resulting from the digestion prior to the
disinfection processing step are preferred. For example, such
processes may include removing only a portion of the water (e.g.
about 10% to about 98% by weight of the water present) prior to the
dialysis/disinfection step, or may include rendering the digest to
a solid by drying the material by lyophilization or otherwise,
reconstituting the dried material in an aqueous medium, and then
treating that aqueous medium with the dialysis/disinfection
step.
[0056] Certain impacts of dialysis processing conditions upon ECM
hydrolysate gels are illustrated in specific work to date described
more particularly in Examples 2-5 below. Generally, several
different submucosa hydrolysates were prepared while varying the
acid present during pepsin digestion and varying the concentration
of ECM hydrolysate present during dialysis against a peracetic acid
(PAA) solution. Specifically, a first gel (A1) was prepared using
0.5 M acetic acid in the pepsin digestion solution, and about 5-15
mg/ml ECM hydrolysate during the PAA disinfection; a second gel
(A2) was prepared using 0.5 M acetic acid in the pepsin digestion
solution, and about 130-150 mg/ml ECM hydrolysate during the PAA
disinfection; a third gel (H1) was prepared using 0.01 M
hydrochloric acid in the pepsin digestion solution, and about 5-15
mg/ml ECM hydrolysate during the PAA disinfection; and a fourth gel
(H2) was prepared using 0.01 M hydrochloric acid in the pepsin
digestion solution, and about 130-150 mg/ml ECM hydrolysate during
the PAA disinfection. The processed ECM hydrolysates were provided
in a solution of 0.1 M HCl at a concentration of about 30 mg/ml,
and then PBS was added and the pH of the mixture was adjusted to
7.5-7.6 with 0.25 M NaOH to gel the composition. The mechanical
properties of the various gels were then assessed. The results are
summarized in Table 1 below.
TABLE-US-00001 TABLE 1 Compressive Compressive Gel Modulus (kPa)
Strength (kPa A1 1 0.5 A2 7 2 H1 10 3 H2 20 7
[0057] As can be seen, the gels prepared using high submucosa
hydrolysate concentrations during the disinfection step (A2,H2)
were relatively stronger than those prepared using low submucosa
hydrolysate concentrations (A1, H1). In addition, in cell growth
assays, the A2 and H2 gels demonstrated an improved capacity to
support the proliferation of primary human dermal fibroblast and
primary human bladder smooth muscle cells as compared to the A1 and
H1 gels. In other observations, the gels prepared from ECM
hydrolysate materials resultant of HCl pepsin digestion were
relatively stronger than the corresponding gels resultant of acetic
acid/pepsin digestion. Thus, the conditions used during the
preparation and processing of ECM hydrolysate materials can be
selected and controlled to modulate the physical and biological
properties of the ultimate ECM gel compositions.
[0058] In one mode of operation, the disinfection of the aqueous
medium containing the ECM hydrolysate can include adding the peroxy
compound or other oxidizing disinfectant directly to the ECM
hydrolysate, for example being included in an aqueous medium used
to reconstitute a dried ECM hydrolysate or being added directly to
an aqueous ECM hydrolysate composition. The disinfectant can then
be allowed to contact the ECM hydrolysate for a sufficient period
of time under suitable conditions (e.g. as described above) to
disinfect the hydrolysate, and then removed from contact with the
hydrolysate. In one embodiment, the oxidizing disinfectant can then
be removed using a dialysis procedure as discussed above. In other
embodiments, the disinfectant can be partially or completely
removed using other techniques such as chromatographic or ion
exchange techniques, or can be partially or completely decomposed
to physiologically acceptable components. For example, when using
an oxidizing disinfectant containing hydrogen peroxide (e.g.
hydrogen peroxide alone or a peracid such as peracetic acid),
hydrogen peroxide can be allowed or caused to decompose to water
and oxygen, for example in some embodiments including the use of
agents that promote the decomposition such as thermal energy or
ionizing radiation, e.g. ultraviolet radiation.
[0059] In another mode of operation, the oxidizing disinfectant can
be delivered into the aqueous medium containing the ECM hydrolysate
by dialysis and processed sufficiently to disinfect the hydrolysate
(e.g. as described above), and then removed using other techniques
such as chromatographic or ion exchange techniques in whole or in
part, or allowed or caused to decompose in whole or in part as
discussed immediately above.
[0060] Peroxygen compounds that may be used in the disinfection
step include, for example, hydrogen peroxide, organic peroxy
compounds, and preferably peracids. Such disinfecting agents are
used in a liquid medium, preferably a solution, having a pH of
about 1.5 to about 10.0, more desirably about 2.0 to about 6.0. As
to peracid compounds that can be used, these include peracetic
acid, perpropioic acid, or perbenzoic acid. Peracetic acid is the
most preferred disinfecting agent for purposes of the present
invention.
[0061] When used, peracetic acid is desirably diluted into about a
2% to about 50% by volume of alcohol solution, preferably ethanol.
The concentration of the peracetic acid may range, for instance,
from about 0.05% by volume to about 1.0% by volume. Most
preferably, the concentration of the peracetic acid is from about
0.1% to about 0.3% by volume. When hydrogen peroxide is used, the
concentration can range from about 0.05% to about 30% by volume.
More desirably the hydrogen peroxide concentration is from about 1%
to about 10% by volume, and most preferably from about 2% to about
5% by volume. The solution may or may not be buffered to a pH from
about 5 to about 9, with more preferred pH's being from about 6 to
about 7.5. These concentrations of hydrogen peroxide can be diluted
in water or in an aqueous solution of about 2% to about 50% by
volume of alcohol, most preferably ethanol. Additional information
concerning preferred peroxy disinfecting agents can be found in
discussions in U.S. Pat. No. 6,206,931, which is herein
incorporated by reference.
[0062] ECM gel materials of the present invention can be prepared
to have desirable properties for handling and use. For example,
fluidized ECM hydrolysates can be prepared in an aqueous medium,
which can thereafter be caused or allowed to form of a gel. Such
prepared aqueous mediums can have any suitable level of ECM
hydrolysate therein for subsequent gel formation. Typically, the
ECM hydrolysate will be present in the aqueous medium to be gelled
at a concentration of about 2 mg/ml to about 200 mg/ml, more
typically about 20 mg/ml to about 200 mg/ml, and in some preferred
embodiments about 30 mg/ml to about 120 mg/ml. In preferred forms,
the aqueous ECM hydrolysate composition to be gelled will have an
injectable character, for example by injection through a needle
having a size in the range of 18 to 31 gauge (internal diameters of
about 0.047 inches to about 0.004 inches).
[0063] Furthermore, gel compositions can be prepared so that in
addition to neutralization, heating to physiologic temperatures
(such as 37.degree. C.) will substantially reduce the gelling time
of the material. As well, once the material is gelled, it can
optionally be dried to form a sponge solid material. It is
contemplated that commercial products may constitute any of the
these forms of the ECM gel composition, e.g. (i) packaged, sterile
powders which can be reconstituted in an acidic medium and
neutralized and potentially heated to form a gel, (ii) packaged,
sterile aqueous compositions including solubilized ECM hydrolysate
components under non-gelling (e.g. acidic) conditions; (iii)
packaged, sterile gel compositions, and (iv) packaged, sterile,
dried sponge compositions; or other suitable forms. In one
embodiment of the invention, a medical kit is provided that
includes a packaged, sterile aqueous composition including
solubilized ECM hydrolysate components under non-gelling (e.g.
acidic) conditions, and a separately packaged, sterile aqueous
neutralizing composition (e.g. containing a buffer and/or base)
that is adapted to neutralize the ECM hydrolysate medium for the
formation of a gel. In another embodiment of the invention, a
medical kit includes a packaged, sterile, dried (e.g. lyophilized)
ECM hydrolysate powder, a separately packaged, sterile aqueous
acidic reconstituting medium, and a separately packaged sterile,
aqueous neutralizing medium. In use, the ECM hydrolysate powder can
be reconstituted with the reconstituting medium to form a
non-gelled mixture, which can then be neutralized with the
neutralizing medium for the formation of the gel.
[0064] Medical kits as described above may also include a device,
such as a syringe, for delivering the neutralized ECM hydrolysate
medium to a patient. In this regard, the sterile, aqueous ECM
hydrolysate medium or the sterile ECM hydrolysate powder of such
kits can be provided packaged in a syringe or other delivery
instrument. In addition, the sterile reconstituting and/or
neutralizing medium can be packaged in a syringe, and means
provided for delivering the contents of the syringe into to another
syringe containing the aqueous ECM hydrolysate medium or the ECM
hydrolysate powder for mixing purposes. In still other forms of the
invention, a self-gelling aqueous ECM hydrolysate composition can
be packaged in a container (e.g. a syringe) and stable against gel
formation during storage. For example, gel formation of such
products can be dependent upon physical conditions such as
temperature or contact with local milieu present at an implantation
site in a patient. Illustratively, an aqueous ECM hydrolysate
composition that does not gel or gels only very slowly at
temperatures below physiologic temperature (about 37.degree. C.)
can be packaged in a syringe or other container and potentially
cooled (including for example frozen) prior to use for injection or
other implantation into a patient.
[0065] In particular applications, ECM hydrolysate compositions
that form hydrogels at or near physiologic pH and temperature will
be preferred for in vivo bulking applications, for example in the
treatment of stress urinary incontinence, gastroesophageal reflux
disease, cosmetic surgery, vesico urethral reflux, anal
incontinence and vocal cord repair. These forms of the submucosa or
other ECM gel have, in addition to collagen, complex extracellular
matrix sugars and varying amounts of growth factors in other
bioactive agents that can serve to remodel tissue at the site of
implantation. These ECM hydrolysate compositions can, for example,
be injected into a patient for these applications.
[0066] ECM gels and dry sponge form materials of the invention
prepared by drying ECM gels can be used, for example, in wound
healing and/or tissue reconstructive applications, or in the
culture of cells.
[0067] Generally, it has been found that the manipulations used to
prepare ECM hydrolysate compositions and gellable or gelled forms
thereof can also have a significant impact upon growth factors or
other ECM components that may contribute to bioactivity. Techniques
for modulating and sampling for levels of FGF-2 or other growth
factors or bioactive substances can also be used in conjunction
with the manufacture of the described ECM hydrolysate compositions
of the invention. Illustratively, it has been discovered that the
dialysis/disinfection processes of the invention employing peroxy
compounds typically cause a reduction in the level of FGF-2 in the
ECM hydrolysate material. In work to date as described in Examples
2-5, such processing using peracetic acid as disinfectant has
caused a reduction in the level of FGF-2 in the range of about 30%
to about 50%. Accordingly, to retain higher levels of FGF-2, one
can process for a minimal about of time necessary to achieve the
desired disinfection of the material; on the other hand, to reduce
the FGF-2 to lower levels, the disinfection processing can be
continued for a longer period of time. In one embodiment of the
invention, the disinfection process and subsequent steps will be
sufficiently conducted to result in a medically sterile aqueous ECM
hydrolysate composition, which can be packaged using sterile
filling operations. In other embodiments, any terminal
sterilization applied to the ECM hydrolysate material (e.g. in
dried powder, non-gelled aqueous medium, gelled or sponge form) can
also be selected and controlled to optimize the level of FGF-2 or
other bioactive substances in the product. Terminal sterilization
methods may include, for example, high or low temperature ethylene
oxide, radiation such as E-beam, gas plasma (e.g. Sterrad), or
hydrogen peroxide vapor processing.
[0068] Preferred, packaged, sterilized ECM hydrolysate products
prepared in accordance with the invention will have an FGF-2 level
(this FGF-2 being provided by the ECM hydrolysate) of about 100
ng/g to about 5000 ng/g based upon the dry weight of the ECM
hydrolysate. More preferably, this value will be about 300 ng/g to
about 4000 ng/g. As will be understood, such FGF-2 levels can be
determined using standard ELISA tests (e.g. using the Quantikine
Human Basic Fibroblast Growth Factor ELISA kit commercially
available from R&D Systems).
[0069] In order to promote a further understanding of the present
invention and its features and advantages, the following specific
examples are provided. It will be understood that these examples
are illustrative and are not limiting of the invention.
EXAMPLE 1
[0070] Small intestinal submucosa material was harvested and
disinfected with peracetic acid as described in U.S. Pat. No.
6,206,931. The submucosa material was lyophilized, packaged in
medical packaging comprised of polyester/Tyvek and sterilized by
various methods including ethylene oxide (EO), gas plasma (hydrogen
peroxide vapor), and E-beam radiation (20 kGy (plus/minus 2 kGy).
The resultant submucosa material was frozen in liquid nitrogen and
ground to a powder. The material was then extracted with an
extraction buffer containing 2M urea, 2.5 mg/ml heparin, and 50 mM
Tris buffer, at pH 7.5 at 4.degree. C. under constant stirring for
24 hours. After 24 hours, the extraction medium was transferred to
centrifuge tubes and the insoluble fraction pelleted at
12000.times.G. The supernatant was transferred to dialysis tubing
(MW cutoff 3500) and dialyzed exhaustively against high purity (18
megaohm) water. Following dialysis the dialysate was centrifuged at
12000.times.G to remove any additional particulate matter and the
resulting soluble extract was lyophilized. Prior to measurement the
extract was reconstituted at 10 mg/dry weight per ml in the
manufacturer-provided diluent (R&D Systems). Samples were
centrifuged to remove any insoluble matter. The resulting
supernatents were recovered and assayed for FGF-2 content using the
Quantikine Human Basic Fibroblast Growth Factor Immunoassay
(R&D Systems). The results are summarized in Table 2 below.
TABLE-US-00002 TABLE 2 ELISA Summary--CBI Growth Extracted Tissues
factor range % of Sterilization (ng/g) Non-sterile* NONE 100-210
100 EO (low temp) 28-50 24 EO (high temp) 18-40 18 E-beam 50-150 66
Gas Plasma 30-125 49 *based upon the average of 8 experiments.
[0071] As can be seen, E-beam and gas plasma sterilization had a
significantly lower impact in reducing the level of extractable,
bioactive FGF-2 in the materials. On the other hand, ethylene oxide
sterilization at both low and high temperatures had a significant
impact in lowering the level of extractable, bioactive FGF-2.
EXAMPLE 2
[0072] Raw (isolated/washed but non-disinfected) porcine small
intestine submucosa was frozen, cut into pieces, and cryoground to
powder with liquid nitrogen. 50 g of the submucosa powder was mixed
with one liter of a digestion solution containing 1 g of pepsin and
0.5 M acetic acid. The digestion process was allowed to continue
for 48-72 hours under constant stirring at 4.degree. C. At the end
of the process, the digest was centrifuged to remove undigested
material. The acetic acid was then removed by dialysis against 0.01
M HCl for approximately 96 hours at 4.degree. C. The resulting
digest was transferred (without concentration) into a semipermeable
membrane with a molecular weight cut off of 3500, and dialyzed for
two hours against a 0.2 percent by volume peracetic acid in a 5
percent by volume aqueous ethanol solution at 4.degree. C. This
step served both to disinfect the submucosa digest and to
fractionate the digest to remove components with molecular weights
below 3500. The PAA-treated digest was then dialysed against 0.01 M
HCl for 48 hours at 4.degree. C. to remove the peracetic acid. The
sterilized digest was concentrated by lyophilization, forming a
material that was reconstituted at about 30 mg/ml solids in 0.01 M
HCl and neutralized with phosphate buffered NaOH to a pH of about
7.5-7.6 and heated to physiologic temperature to form a submucosa
gel.
EXAMPLE 3
[0073] A second acetic acid processed submucosa gel was made using
a process similar to that described in Example 2 above, except
concentrating the digest prior to the PAA treatment. Specifically,
immediately following the removal of acetic acid by dialysis, the
digest was lyophilized to dryness. A concentrated paste of the
digest was made by dissolving a pre-weighed amount of the
lyophilized product in a known amount of 0.01 M HCl to prepare a
mixture having an ECM solids concentration of about 50 mg/ml. The
concentrated paste was then dialysed against the PAA solution for 2
hours and then against 0.01 M HCl for removal of PAA in the same
manner described in Example 2. The digest was adjusted to about 30
mg/ml solids and neutralized with phosphate buffered NaOH to a pH
of about 7.5-7.6 and heated to physiologic temperature to form a
submucosa gel.
EXAMPLE 4
[0074] An HCl processed submucosa gel was made using a procedure
similar to that described in Example 2, except using 0.01 M of HCl
in the pepsin/digestion solution rather than the 0.5 M of acetic
acid, and omitting the step involving removal of acetic acid since
none was present. The digest was used to form a gel as described in
Example 2.
EXAMPLE 5
[0075] Another HCl processed submucosa gel was made using a
procedure similar to that described in Example 3, except using 0.01
M of HCl in the pepsin/digestion solution rather than the 0.5 M of
acetic acid, and omitting the step involving removal of acetic acid
since none was present. The digest was used to form a gel as
described in Example 3.
[0076] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same is to be
considered as illustrative and not restrictive in character, it
being understood that only the preferred embodiment has been shown
and described and that all changes and modifications that come
within the spirit of the invention are desired to be protected. In
addition, all publications cited in this application are indicative
of the abilities possessed by those of ordinary skill in the
pertinent art and are hereby incorporated by reference in their
entirety as if each had been individually incorporated by reference
and fully set forth.
* * * * *