U.S. patent application number 10/722034 was filed with the patent office on 2004-06-03 for resorbable implant materials.
Invention is credited to Desmith, Amy, Francis, Ralph T., Oray, B. Nicholas, Zhao, Qing Hong.
Application Number | 20040107006 10/722034 |
Document ID | / |
Family ID | 23566590 |
Filed Date | 2004-06-03 |
United States Patent
Application |
20040107006 |
Kind Code |
A1 |
Francis, Ralph T. ; et
al. |
June 3, 2004 |
Resorbable implant materials
Abstract
A non-crosslinked, decellularized and purified mammalian tissue
(e.g., bovine pericardium) having particular use as an implantable
resorbable material. The material is treated by alkylating its
primary amine groups in a manner sufficient to reduce the
antigenicity of the tissue, permitting the treated tissue to be
used in vivo and without crosslinking, and in turn, permitting it
to be resorbable. The material can be used in surgical repair of
soft tissue deficiencies for a certain period of time while the
implant itself is gradually remodeled or absorbed by the host. Also
provided are a method of preparing such a material, as well as a
method of using such a material for surgical repair.
Inventors: |
Francis, Ralph T.; (New
Brighton, MN) ; Zhao, Qing Hong; (Andover, MN)
; Desmith, Amy; (Circle Pines, MN) ; Oray, B.
Nicholas; (Woodbury, MN) |
Correspondence
Address: |
INTELLECTUAL PROPERTY GROUP
FREDRIKSON & BYRON, P.A.
4000 PILLSBURY CENTER
200 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Family ID: |
23566590 |
Appl. No.: |
10/722034 |
Filed: |
November 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10722034 |
Nov 24, 2003 |
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10099425 |
Mar 14, 2002 |
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6652594 |
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10099425 |
Mar 14, 2002 |
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PCT/US00/25234 |
Sep 14, 2000 |
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PCT/US00/25234 |
Sep 14, 2000 |
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09396279 |
Sep 15, 1999 |
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6312474 |
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Current U.S.
Class: |
623/23.72 ;
8/94.11 |
Current CPC
Class: |
A61L 31/044 20130101;
A61L 27/3629 20130101; A61L 27/3687 20130101; A61L 31/043 20130101;
A61L 27/3604 20130101; A61L 27/24 20130101; A61L 27/362 20130101;
A61L 27/227 20130101 |
Class at
Publication: |
623/023.72 ;
008/094.11 |
International
Class: |
A61F 002/02 |
Claims
What is claimed is:
1. A resorbable, remodelable implant material comprising a sterile,
non-crosslinked, decellularized and purified mammalian tissue
having a major percentage of its available amine groups
alkylated.
2. A material according to claim 1 wherein the tissue is selected
from the group consisting of serous and fibro-serous membranes.
3. A material according to claim 2 wherein the tissue is selected
from the group consisting of pericardium, peritoneum, fascia lata,
dura mater, dermis and small intestinal submucosa.
4. A material according to claim 3 wherein the tissue comprises
bovine pericardium.
5. A material according to claim 1 wherein the material has been
alkylated by an alkylating agent selected from the group consisting
of 1,2-epoxy-R compounds where R is an alkyl group up to 6 carbon
atoms.
6. A material according to claim 5 wherein the alkylating agent is
propylene oxide.
7. A material according to claim 1 wherein the alkylating agent is
methyl glycidyl ether.
8. A material according to claim 1 wherein the material is provided
in the form of flat or textured sheets or strips.
9. A material according to claim 1 wherein the material is adapted
for use in a surgical application selected from the group
consisting of duraplasty, thoracic, abdominal, urological,
opthalmological, cardiac, and vascular surgery.
10. A process of preparing a resorbable, remodelable implant
material according to claim 1, the method comprising the step of
treating a biological tissue with an alkylating agent under
conditions suitable to alkylate a major percentage of available
amine groups in the tissue, and sterilizing the treated tissue for
use in vivo.
11. A process according to claim 10 wherein the cleaned tissue is
treated with a base prior to the alkylating step.
12. A process according to claim 10 wherein the alkylating agent is
used at a pH of between about 9 and about 11.
13. A process according to claim 10 wherein the concentration of
alkylating agent is between about 2% (v/v) and about 5% (v/v).
14. A process according to claim 10 wherein the tissue is exposed
to the alkylating agent for at least 48 hours.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of patent
application having U.S. Ser. No. 10/099,425, filed 14 Mar. 2002,
which is a continuation of and claims priority to International
Application No. PCT/US00/25234 (published as International
Publication No. WO 01/19423), filed 14 Sep. 2000 and designating
the United States, which in turn claims priority from patent
application having U.S. Ser. No. 09/396,279, filed 15 Sep. 1999,
the entire disclosure of which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The invention relates to materials for the use as implants
within the body, and in particular, to resorbable and remodelable
materials for such use.
BACKGROUND OF THE INVENTION
[0003] Various resorbable (occasionally referred to as absorbable
or "remodelable") materials presently exist for use in prosthetic
applications, e.g., as patches, implants and/or as components of
prosthetic devices.
[0004] Synthetic resorbable materials made from the polyesters,
polylactide and polyglycolide, for example, have found use in
various fields of medicine (See, e.g., Ashammaki, N. A., J. Biomed.
Mater. Res., 33: 297-303; 1996). Versions of these materials exist
commercially under the tradenames Vicryl.RTM. (Ethicon, Inc.) and
Dexon.RTM. (Davis & Geck, Inc.). The gradual decomposition of
these polymers is facilitated by hydrolysis, and catalyzed by
biochemical action of the host tissues (Hanbrough, J. F., et al.,
J. Burn Care Rehab., 14: 485-494; 1993). These materials may be
produced as membranes or as woven mesh in the case of producing
resorbable suture.
[0005] While synthetic resorbable materials are a rather recent
phenomenon, collagenous materials have been used as prosthetic
grafting for many years; as in the case of lyophilized human dura,
dating back to 1954. As a common practice for several years, such
collagenous materials have been crosslinked with an agent such as
glutaraldehyde, in order to diminish the antigenicity of a
xenograft while increasing its resistance to enzymatic degradation
produced by host tissue responses (Gratzer, P. F., et al., J.
Biomed. Mater. Res., 31: 533-543; 1996). Polyepoxy compounds have
also been used for such purposes, however are more stable with
regard to the resulting alkylated amines in the collagen (Sung,
H-W., et al., J. Biomater. Sci. Polymer Edn., 8: 587-600; 1997).
While crosslinked tissues work well as long-term implants, they are
not resorbable and as such, do not promote host tissue remodeling,
or in turn, the eventual replacement of a graft by the body
itself.
[0006] Aesculap AG & Co. (B. Braun Surgical) offers products
under the tradename Lyoplant.RTM., in the form of a bovine
pericardium-based resorbable replacement for dura mater.
Lyoplant.RTM. is produced by a process that involves mechanical
removal of adherent fat and connective tissue, chemical treatment
to inactivate enzymes and potential pathogens, freeze-drying,
cutting to various sizes, packaging and terminal sterilization with
ethylene oxide. The product is indicated to be used for covering
cerebral and cerebellar dural defects, for decompressive duraplasty
in cases of increased intracranial pressure, for covering spinal
dural defects and for spinal decompressive duraplasty. This
material has been observed to be fully remodeled within one year
after implant.
[0007] Tutogen Medical, Inc. provides processed pericardium
products under the tradename Tutoplast.RTM., in the form of a
solvent-dehydrated, gamma-irradiated preserved human pericardium.
The processing of Tutoplast.RTM. tissue involves thorough cleaning,
processing, dehydration and preservation. The process is said to
leave no deleterious residue and minimizes antigenic potential.
Collagenous connective tissue with multidirectional fibers retains
the mechanical strength and elasticity of native pericardium, while
providing the basic formative structure to support replacement by
new endogenous tissue. This tissue is indicated for use in a
variety of surgical applications, including duraplasty (as a
substitute for human dura mater), and in abdominal, urological,
opthalmological, and vascular surgery. The absorption process and
reformation of endogenous tissue begins one to two days after
implantation and continues for weeks, months, or years, depending
on the size of the graft and the responsiveness of the graft site.
Mentor Corporation has entered a strategic alliance with Tutogen
Medical, Inc., to use the Tutoplast.RTM. technology to manufacture
resorbable slings for urinary incontinence (Suspend.TM.).
[0008] A variety of other uses of resorbable materials are
described in the patent literature. See, for instance, U.S. Pat.
No. 5,895,420 (Mirsch, II, et al., "Bioresorbable Heart Valve
Support"), which, relates to bioprosthetic heart valve stents that
are fashioned of resorbable materials. Such stents may be
configured as sheaths or frames contoured to the shape of a
valvular graft. The stents are eventually resorbed by the patient,
leaving a functional "stentless" valve with improved hemodynamic
characteristics compared to stented valve implants.
[0009] Various other resorbable materials have been suggested or
proposed for use with vascular of non-vascular implants. For
example, Goldberg et al., U.S. Pat. No. 5,085,629 discloses a
biodegradable infusion stent for use in treating ureteral
obstructions. Stack, et al., U.S. Pat. No. 5,306,286 discloses an
absorbable stent for placement within a blood vessel during
coronary angioplasty. Duran, U.S. Pat. No. 5,376,112 discloses an
annuloplasty ring to be implanted into the heart to function
together with the native heart valve.
[0010] In another aspect, U.S. Pat. No. 5,837,278 (Geistlich, et
al., "Resorbable Collagen Membrane for Use in Guided Tissue
Regeneration"), describes the use of a collagen-containing membrane
in guided tissue regeneration. The patent provides a resorbable
collagen membrane for use in guided tissue regeneration wherein one
face of the membrane is fibrous thereby allowing cell growth
thereon and the opposite face of the membrane is smooth, thereby
inhibiting cell adhesion thereon.
[0011] Finally, see U.S. Pat. No. 5,413,798 (Scholl, et al.) which
describes a process for treating bovine pericardial tissue to
increase resistance to biological degradation by wet-chemical
processing. The use of the tissue is exemplified in the form of an
implant which, after three and six months post implantation, was
well integrated so that it was no longer distinguishable from
autochthonous dura (revitalized by fibrocytes and traversed by
blood vessels in the marginal zones). The inner side of the implant
is coated with the same cell type as the autologous dura.
[0012] On yet another topic, certain articles describe basic
research directed to studying the effect of alkylating agents on
materials such as collagen. See, for example, Sung, H. W., et al.,
J. Biomed. Mater. Res. 37:376-383 (1997) and Tu, R. et al., J.
Biomed. Mater. Res., 28:677-684 (1994). To the best of Applicants'
knowledge, however, no such reference suggests the manner in which
such materials might be used in vivo, nor in turn, do they address
the question of whether such materials can be tolerated, let alone
resorbed and remodeled, by the body.
[0013] The present assignee is recognized as a leader in the
development and manufacture of pericardium based materials. See,
for instance, U.S. Pat. Nos. 5,752,965; 5,575,803; 5,549,628;
5,503,638; and 4,915,113 and International Application No.
US98/25674, the disclosures of each of which are incorporated
herein by reference. Generally, the pericardium materials are
crosslinked, e.g., using glutaraldehyde, and hence are typically
considered non-resorbable. Such materials have been used in a
variety of applications, including as patches, suture and staple
line buttress members, and pledgets.
SUMMARY OF THE INVENTION
[0014] The present invention provides a non-crosslinked,
decellularized and purified mammalian tissue (e.g., bovine
pericardium) having particular use as an implantable material in a
manner that is both resorbable and remodelable. The material is
prepared by alkylating the primary amine groups of natural tissue
in a manner sufficient to reduce the antigenicity of the tissue,
and in turn, to an extent that permits the treated tissue to be
used in vivo and without crosslinking, thereby permitting it to be
resorbable.
[0015] The material can be used, for instance, in surgical repair
of soft tissue deficiencies, for a period of time, while the
implant itself is gradually remodeled or absorbed by the host. In a
related aspect, the invention provides a method of preparing such a
material, as well as a method of using such a material for surgical
repair. As used herein with respect to a material of the present
invention, the word "resorb" and inflections thereof will refer to
a material that, once implanted in vivo, is absorbed by the body
over time and without undue deleterious effects on or within the
body itself. The word "remodel" and inflections thereof, as used
with regard to a material of the present invention, will refer to a
resorbable material that is adapted, e.g., by virtue of its
location and method of implantation within the body, to encourage
and/or permit the body to replace some or all of the structure
and/or function of the implant with newly formed natural tissue.
While not intending to be bound by theory, at least in some
embodiments of the present invention, remodeling appears to occur
by gradual bodily processes in which substantial portions of the
implant material are gradually resorbed, while an inherent fibrous
network of the implant is retained at the site. The network, in
turn, is used by the body as essentially scaffolding for the
generation of new tissue or tissue components.
[0016] In a preferred embodiment, the invention provides a
resorbable implantable material comprising a non-crosslinked,
decellularized and purified mammalian tissue having most of its
free amine groups alkylated. In a particularly preferred
embodiment, the tissue is selected from the group consisting of
pericardium, peritoneum, fascia lata, dura mater, dermis and small
intestinal submucosa, and the material has been alkylated by an
alkylating agent selected from the group consisting of 1,2-epoxy-R
compounds where R is an alkyl group up to 6 carbon atoms. Such a
material can be provided in any suitable form, e.g., as flat or
textured sheets or strips, and can be adapted for use in a variety
of surgical applications, including those selected from the group
consisting of duraplasty, thoracic, abdominal, urological,
opthalmological, cardiac, and vascular surgery.
DETAILED DESCRIPTION
[0017] A tissue of the present invention can be obtained from any
suitable source including mammalian sources, e.g., in the form of
collagenous connective tissue with three dimensional intertwined
fibers. Such tissues generally include serous and fibro-serous
membranes. In a particularly preferred embodiment, the tissue
source is selected from bovine pericardium, peritoneum, fascia
lata, dura mater, dermis, and small intestinal submucosa. In a
further preferred embodiment, the tissue is bovine pericardium, and
is treated using a method as described herein to provide the
treated tissue with an optimal combination of biocompatability,
thickness, and other physical and physiological properties.
[0018] Tissues of the present invention can be provided from dura
mater, for instance, for use in neurosurgical applications.
Collagenous connective tissue with three dimensional intertwined
fibers, when treated in the manner described herein, retains the
multidirectional and mechanical strength of native dura matter,
while providing the basic formative structure to support
replacement by new endogenous tissue.
[0019] While it is desirable to reduce or eliminate antigenic
properties of xenografic or even allografic tissue-based material
to be implanted into a body, if the body's absorption and/or
remodeling of the material are desired, crosslinking cannot be
performed. In order to specifically perform such modification of a
collagen-based material, a monofunctional reagent is therefore
used. The reagent is "monofunctional" in that it is adapted to
react with, and therefore terminate or "cap" the available amine
functionalities of tissue proteins, but will not further react with
adjacent groups. An optimal reagent of this invention, therefore,
is preferably a relatively small and structurally simple compound
that, upon reaction with protein groups such as amines, will bind
to those groups but will not otherwise alter the biological
properties of the collagen matrix to an extent that renders the
tissue unsuitable for its intended use.
[0020] In a particularly preferred embodiment, a tissue of the
present invention is treated by a process that includes alkylating
a major percentage of its available amine groups to an extent
sufficient to permit the tissue to be implanted and used in vivo.
Preferably a tissue is processed by alkylating its amines to an
extent sufficient to react 80% or more, preferably 90% or more, and
most preferably 95% or more of the amine groups originally present.
The efficacy and extent of alkylation can be determined by a
variety of means, as described herein, including the use of a
ninhydrin-based assay ("amine index") to determine a comparative
level of amine groups, before and after treatment (see, e.g., Sung
H-W, et al. Art Org., 21: 50-58; 1997. Sung, H-W, et al., J.
Biomed. Mater. Res. 33: 177-186. 1996). Preferably the efficacy and
extent of the alkylation process is further assessed by determining
unreacted amounts in the batch incubation of the alkylating agent
used.
[0021] Preferred alkylating agents can be used, for instance, at a
pH of between about 9 and about 11, and at a concentration of
between about 2% (v/v) and about 5% (v/v), by exposing the tissue
to a solution containing the agent for at least 48 hours.
[0022] Preferred alkylating agents include small and reactive amine
alkylating agents, such as formaldehyde, and 1,2-epoxy compounds.
The epoxy agents offer an advantage over formaldehyde in that they
tend to produce more stable adducts in their reactions with amines
(Sung, H-W., et al., Biomater., 17: 2357-2365; 1996). 1,2-epoxy
agents can react with a primary amine at alkaline pH to produce an
extremely stable 2-hydroxy secondary amine. However, an aldehyde
such as formaldehyde reacts with a primary amine to produce a
marginally unstable, reversible double-bonded aldimine (Girardot,
J-M. and Girardot, M-N., J. Heart Valve Dis., 5: 518-525;
1996).
[0023] Of the various monofunctional 1,2-epoxy agents, propylene
oxide ("PO") is particularly preferred since it possesses
properties that render its inclusion into a material process
simple, yet effective. Propylene oxide (epoxypropane) has been used
for several years as a sterilant, mostly in a gaseous state,
although at room temperature, it exists as a liquid (Hart, A. and
Brown, W., Appl. Microbiol., 28: 1069-1070; 1975). Many years ago,
PO was revealed to directly modify carboxylic, thiol, phenolic and
amine groups of proteins under certain conditions (Fraenkal-Conrat,
H., J. Biol. Chem., 154: 227-238; 1944). As has been demonstrated
with other epoxides, propylene oxide reacts predominantly with
amines at alkaline pH. Collagen swells at alkaline pH rendering it
more accessible to be alkylated with a water-soluble agent such as
propylene oxide.
[0024] Another preferred monofunctional epoxy reagent for use in
the present invention is methyl glycidyl ether, as is produced by
the Nagase Corp. of Osaka, Japan and sold under the product name
Denacol.RTM. EX-131. This product has a low molecular weight, is
water-soluble and was shown to be a more potent alkylator of
porcine pericardium than formaldehyde (Sung, H-W., et al., J.
Biomed. Mater. Res., 35: 147-155; 1997).
[0025] In addition to the "amine index", another test may be used
to confirm tissue modification by an amine alkylating agent. The
denaturation (shrink) temperature (T.sub.d) is often used to verify
the crosslinking of collagen by an agent such as glutaraldehyde. It
is typically observed that upon chemical crosslinking, the T.sub.d
increases significantly, apparently due to increased stabilization
of the hydrogen bonds present in the collagen. In contrast, upon
alkylation with a monofunctional agent such as propylene oxide, the
T.sub.d decreases significantly. This phenomenon is believed to
occur due to branching of the collagen polymer by the action of the
alkylating agent and the subsequent alteration of the collagen
matrix (Tu, R., et al., J. Biomed. Mater. Res., 28: 677-684;
1994).
[0026] In a preferred embodiment, a tissue of the present invention
is also treated with a base such as sodium hydroxide (NaOH), in
order to further lesson the already minimal possibility of Bovine
Spongiform Encephalopathy (BSE) transmission. Histological analyses
of NaOH-treated tissue (pericardium, for example) reveals virtually
complete decellularization due to this treatment. Since the
cellular component of tissue is known to contain the vast majority
of the antigen load (Courtman, D. W., et al., J. Biomed. Mater.
Res., 28: 655-666; 1994), decellularization treatment with NaOH can
complement the use of an alkylating agent in reducing
antigenicity.
[0027] A tissue of the present invention can be used to fabricate a
prosthetic article having any suitable shape or configuration, and
in any suitable dimensions for its intended use. For instance, the
tissue can be provided and packaged in a flat configuration (e.g.,
sheet or tape-like), with either or both major surfaces thereof
being optimally textured or modified (e.g., by the covalent
attachment, entrapment, and/or adsorption of biologically active
factors, lubricious agents, antimicrobial agents and the like).
[0028] In a preferred embodiment, a process of the present
invention includes the steps of:
[0029] a) obtaining pericardium from a suitable (e.g.,
USDA-approved) source,
[0030] b) cleaning the tissue and optionally, and preferably,
treating the tissue, e.g., in order to decellularize it and/or to
reduce/eliminate potential BSE infectivity,
[0031] c) alkylating the tissue (e.g., hydroxypropylation using
propylene oxide) to cap a major percentage of available (e.g.,
potentially reactive) amine groups, and optionally,
[0032] d) final processing, including one or more of the following
steps: washing, drying, sterilizing and packaging the tissue.
[0033] Natural tissues suitable for use in the process of this
invention preferably meet stringent specifications during donor
screening and laboratory testing to reduce the risk of transmitting
infectious disease. Processing of tissue involves a strict,
quality-controlled procedure, which involves thorough cleaning,
processing, dehydration and preservation. The process leaves no
deleterious residue and minimizes antigenic potential.
Sterilization is preferably achieved with the use of gamma or
electron beam radiation (typically 2.5 Mrad) or ethylene oxide
gas.
[0034] A treated tissue of the present invention is indicated for
implantation with a spectrum of indications. Collagenous connective
tissue of this sort, having multidirectional fibers, is able to
retain a substantial amount of the mechanical strength and
elasticity of native pericardium, while providing the basic
formative structure in situ to support replacement by new
endogenous tissue. This tissue is indicated for use in a variety of
surgical applications, including duraplasty (as a substitute for
human dura mater), and in thoracic, abdominal, urological,
opthalmological, cardiac and vascular surgery.
[0035] Implantation should be avoided into areas with active or
latent infection or signs of tissue necrosis, as well as into areas
with compromised circulation or in any disorder that would create
an unacceptable risk of post-operative complications.
[0036] The tissue can be packaged using conventional means, such
that the tissue and package contents remain sterile and
non-pyrogenic as long as the package is not opened and/or damaged.
The graft must be used before the expiration date. Those skilled in
the appropriate art will appreciate the manner in which appropriate
placement and fixation of the tissue in situ can be critical
factors in avoiding potentially adverse effects on the graft
service life. A tissue of this invention can be prepared and
packaged in various sizes (e.g., thickness, length and width). The
dimensions of tissue used should correspond to the size of the
respective defect.
[0037] Once implanted, the absorption process and reformation of
endogenous tissue begins one to two days after implantation and
continues for weeks, months, or years, depending on the size of the
graft and the responsiveness of the graft site. It is recommended
that, if packaged in a dry or dehydrated condition, the tissue be
rehydrated prior to use for about 2 to about 30 minutes, depending
on the consistency desired, using aseptic/sterile technique. The
surgeon should also monitor the effect of rehydration by visual
inspection, both in the course of rehydration and while cutting and
shaping the graft. Implantation should be performed in such a way
that the free edges of the implant do not extend into areas where
the possibility of adhesion may present a problem.
[0038] Absorbable or nonabsorbable suture material, glue, etc. can
be used to fix the tissue in place. For a continuous suture,
absorbable suture material and round atraumatic needles are
recommended, while suture gauge depends on the surgical indication.
The suture should be located two to three millimeters from the edge
of the graft. Better results are obtained by doubling the section
at suture sites that are under moderate to high stress.
[0039] Tissues of the present invention provide a variety of
features and advantages, including the fact that they are
immediately available for surgery and can save valuable operating
room time. Moreover, there is no secondary surgery site and less
stress for the patient; which can result in less time under
anesthesia, no donor site pain or morbidity, and less cost. Since
the tissues can be made available in a wide range of sizes, the
surgeon can choose the size needed, leading to minimal waste. As
with all biological products, it is not possible to provide an
absolute guarantee of freedom from contaminating infectious
diseases such as hepatitis, Creutzfeld-Jakob Disease (CJD) or
Bovine Spongiform Encephalopathy (BSE). Processing treatments, such
as the use of NaOH in the cases of CJD and BSE, have shown to be
capable of reducing the risk of any transmission, and are
particularly useful in combination with strict donor screening and
laboratory testing. Treated tissues of the present invention can be
stored in a clean, dry environment and at controlled temperatures
between 4.degree. C. and 30.degree. C. (59.degree. to 86.degree.
F.).
TEST PROCEDURES
[0040] Collagenase Assay
[0041] The enzyme class referred to as collagenase has been used
for several years in studying its effects on collagenous
biomaterials. Bacterial collagenase, e.g., from Clostridium
histolyticum, can be used as an accurate predictor of the
propensity and rate of resorption of a material by a mammalian host
(Yannas, I. V., et al., J. Biomed. Mater. Res., 9: 623-628; 1975).
Since modification of collagen by a crosslinking agent results in
greatly diminished susceptibility to the action of collagenase, it
is important that such modification not be performed on tissue to
be resorbed. The mechanism by which crosslinking hinders the
activity of collagenase is not completely understood. Surprisingly,
applicants have found that bacterial collagenase is in fact able to
degrade treated (alkylated) tissues of the present invention. Thus,
tissue alkylated by an agent such as PO possesses pertinent and
functional properties, and the collagenase assay remains a useful
tool for confirming the utility of thus-treated tissue.
[0042] The collagenase assay is a ninhydrin-based assay for the
indication of soluble collagen peptides produced by the action of
the collagenase enzyme, and can be performed as follows:
[0043] 1. Weigh out tissue in the range of 25-30 milligrams.
[0044] 2. Add 3.0 milliliters of collagenase solution [0.01 mg/ml
Collagenase enzyme (Sigma, type 1A) in 50 mM
N-tris[hydroxymethyl]methyl-- 2 aminoethane sulfonic acid ("TES")
buffer with 25 mM calcium chloride, pH 7.4-7.5].
[0045] 3. Incubate at 37.degree. C. for 24 to 96 hours.
[0046] 4. At allotted timepoints, incubate 100 .mu.l of collagenase
solution and 1.0 ml ninhydrin solution [one part 4% (w/v) ninhydrin
in ethylene glycol monoethyl ether to one part 200 mM citric acid,
0.16% (w/v) stannous chloride, pH 5.0] at 95-100.degree. C. for 30
minutes.
[0047] 5. Cool tubes at room temperature.
[0048] 6. Add 250 .mu.l of collagenase sample to 1.0 ml 50%
isopropanol.
[0049] 7. Vortex and read absorbance at 570 nm.
[0050] 8. The absorbance at 570 nm is divided by the weight of the
piece of tissue to give the OD/mg. The OD/mg is the value for the
amount of collagen peptides that has been degraded by the action of
the collagenase enzyme.
[0051] The results of the collagenase assay are determined by
comparing the sample with both positive (untreated) and negative
(glutaraldehyde crosslinked) control samples.
[0052] Amine Index
[0053] The amine index can be defined as the percentage of
initially available amines that have been modified (and thereby
rendered substantially nonreactive in vivo) by reaction with amine
reagents. Such modification will render the amine unable to produce
"Ruhemann's purple" when introduced to ninhydrin, and the relevant
assay can be performed as follows:
[0054] 1. 200 .mu.l of DI water were added to 25-30 milligrams of
tissue.
[0055] 2. Add one milliliter of ninhydrin solution to each
tube.
[0056] 3. Incubate tubes at 95-100.degree. C. for 30-35
minutes.
[0057] 4. Cool tubes at room temperature.
[0058] 5. Add 250 .mu.l of sample to one milliliter of 50%
isopropanol solution.
[0059] 6. Vortex and read absorbance at 570 nm.
[0060] 7. The amine index is calculated.
[0061] In order to calculate the percentage of original amines
modified, the following formula is used: 1 Amine Index ( % ) = [ *
Control ( OD / mg ) - Sample ( OD / mg ) ] Control ( OD / mg )
.times. 100
[0062] The OD/mg is found by dividing the OD @ 570 by the weight of
the piece of tissue. * The control is unmodified tissue.
[0063] Assay for Quantitation of Unreacted Alkylating Agent
[0064] The purpose of this assay is to confirm that although 100%
amine alkylation is typically not attained, it is not due to the
lack of adequate alkylating agent. In essence, this assay is used
to confirm that detectable levels of alkylating agent remain in the
incubation solution upon exhaustive exposure to the tissue. Upon
exposure of tissue to an alkylating agent, the agent solution can
be sampled in order to quantitate the percentage remaining. This
test is in part performed for the purpose of assessing the
efficiency of alkylation. Quantitation is assessed using a standard
curve.
[0065] 1. 10 mM Glycine solution is prepared by adding 0.0375 grams
of glycine to 50 milliliters of 0.2 M carbonate (Na.sup.+2)
buffer.
[0066] 2. Propylene oxide (PO) standards are prepared (e.g.,
ranging from 0.5% PO to 5% PO). The standards are prepared by
adding the correct amount of PO to the carbonate buffer for a total
of five milliliters.
[0067] 3. Add 1 milliliter of the glycine solution to labeled test
tubes.
[0068] 4. Add 1 milliliter of each PO standard to the labeled test
tube.
[0069] 5. Vortex to mix and allow to react for 24 hours at room
temperature.
[0070] 6. After 24 hours, 50 .mu.l of each standard was added to
one milliliter of ninhydrin solution.
[0071] 7. Incubate tubes at 95-100.degree. C. for 30 minutes.
[0072] 8. Cool tubes at room temperature.
[0073] 9. Add 250 .mu.l of standard to one milliliter of 50%
isopropanol solution.
[0074] 10. Vortex and read absorbance at 570 nm.
[0075] The samples containing unknown propylene oxide
concentrations are assessed using the method above. Once the
propylene oxide standard curve is plotted, the samples containing
unknown propylene oxide concentrations can be estimated using the
standard curve.
[0076] Moisture Content
[0077] Moisture content was analyzed on a Mettler-Toledo HG53
Halogen Moisture Analyzer. A temperature setting of 200.degree. C.
was used. Results are recorded in % moisture content.
[0078] Denaturation (Shrink) Temperature
[0079] Denaturation temperature is the temperature at which the
collagen denatures. The test was performed on the ChemDyne MC1000
tensile testing system. The denaturation temperature was measured
using a 30 gram preload in a bath of water at steadily increasing
temperature. Results are expressed in .degree. C.
[0080] The invention will be further described with reference to
the following non-limiting Examples. It will be apparent to those
skilled in the art that many changes can be made in the embodiments
described without departing from the scope of the present
invention. Thus the scope of the present invention should not be
limited to the embodiments described in this application, but only
by embodiments described by the language of the claims and the
equivalents of those embodiments.
EXAMPLE 1
[0081] Bovine pericardial sacs were harvested from USDA inspected
healthy cows, minimum age of 12 months. Fresh pericardium was
obtained and sent through a series of rinses, followed by a final
ice cold water rinse. The tissue was cleaned of extraneous tissue,
and used fresh or stored at -20.degree. C. The following general
procedures were used to prepare treated tissue according to the
present invention.
[0082] All test procedures are performed at 20-25.degree. C.
[0083] NaOH & Neutralization
[0084] 1. Weigh out 40-45 grams of bovine pericardium.
[0085] 2. Place pericardium into one liter of 1.0 M NaOH (40 grams
of NaOH in one liter of DI water) for 60-65 minutes. Take a sample
for pH measurement at the end of soak.
[0086] 3. Decant NaOH; gently squeeze tissue and place in two
liters of filtered DI water for 15-20 minutes.
[0087] 4. Decant DI water and place tissue in two liters of citrate
buffer (28 grams of sodium citrate and 2.0 grams of citric acid in
two liters of DI water) for 60-65 minutes. Take a sample for pH
measurement at the end of soak.
[0088] 5. Decant citrate buffer, gently squeeze tissue and place
tissue in another two liters of DI water for 30-35 minutes.
[0089] Alkylation of Tissue
[0090] 1. Prepare 5% Propylene Oxide solution (50 milliliters of
propylene oxide in 950 milliliters of 0.2 M carbonate buffer, pH
10.5-10.6).
[0091] 2. Place NaOH-treated tissue in propylene oxide
solution.
[0092] 3. Mix on platform shaker for 72-96 hours.
[0093] 4. Remove tissue from solution and place in 1.5 liters of DI
water for 24 hours.
[0094] After hydroxypropylation of tissue, the amine index and the
quantitation of unreacted alkylating agent assays are performed to
verify sufficient alkylation and PO. The tissue was transferred
onto wire mesh racks and dried in a Virtis Genesis vacuum dryer at
115 mtorr.
RESULTS
Table 1
Collagenase Activity
[0095] The table below provides the results of a collagenase assay
when resorbable tissue prepared in the manner described herein was
incubated in 0.01 mg/ml collagenase for 24-96 hours.
1TABLE 1 Tissue 24 hr OD/mg 48 hr OD/mg 72 hr OD/mg 96 hr OD/mg
Untreated 0.319 0.423 0.459 0.481 NaOH 0.502 0.689 0.784 0.874
NaOH/PO 0.674 0.684 0.822 0.973
[0096] OD/mg is the relative value for the amount of collagen that
has been degraded by the action of the collagenase enzyme. It can
be seen that each of the tissues, including the alkylated tissue of
this invention, are susceptible to collagenase digestion,
indicating the likelihood that they would be resorbed within the
body.
2TABLE 2 pH Dependence The following table provides the amine index
results of NaOH/PO- treated tissue when incubated in a 5% PO
solution at two different pHs. Tissue Amine Index @ pH 9.5 Amine
Index @ pH 10.5 NaOH/PO 84.76% 96.29%
[0097] It can be seen that, under the experimental circumstances
involved, the extent of alkylation could be increased at higher
pH.
3TABLE 3 Time Dependence The following table provides the amine
index results of NaOH/PO- treated tissue when incubated in a 5% PO
solution for a period of time at a pH of 10.5. Tissue 72 hr PO
incubation 96 hr PO incubation NaOH/PO 93.95% 96.29%
[0098] It can be seen that there is a slight increase in
alkylation, even in the period from 72 to 96 hour incubation.
4TABLE 4 Assay to Quantitate Unreacted Alkylating Agent The table
below is an example of a standard curve from the assay for
unreacted alkylating agent. 0% 0.5% 1% 2% 3% 4% 5% OD @ 570 0.919
0.707 0.556 0.363 0.274 0.241 0.088 OD @ 570 Unreacted 0.7734
Alkylating agent
[0099] When the data above is plotted it provides a standard curve,
and in a typical preparation, it can be estimated that under the
experimental conditions of this example, between 0.4 and 0.5%
propylene oxide remains unreacted in the alkylation solution.
5TABLE 5 Moisture Content The table below shows how the moisture
content tends to increase as the tissue goes through the alkylation
process (between 72-96 hours). Tissue Moisture Content (%)
Untreated 78.35 NaOH 87.33 NaOH/PO 89.28
[0100]
6TABLE 6 Denaturation Temperature The table below indicates the
manner in which the denaturation (shrink) temperature tends to
decrease as the tissue goes through the alkylation process. Tissue
Denaturation temperature Untreated 65.1.degree. C. NaOH
62.4.degree. C. NaOH/PO 49.2.degree. C.
EXAMPLE 2
In Vivo Biocompatibility and Biostability Study of PO-Capped Bovine
Pericardium
[0101] In this study, propylene oxide (PO) capped, non-crosslinked
bovine pericardium was compared with glutaraldehyde (GA)
crosslinked bovine pericardium in a subcutaneous animal model, in
terms of inflammation, changes in physical property, and remodeling
of implant matrix with the host tissue.
[0102] Preparation of PO Treated Tissue
[0103] Patches (approximately 4 cm.times.6 cm) of fresh bovine
pericardium were first treated in 1N NaOH for one hour, followed by
immersion 2-3 times in 4L of 50 mM citrate buffer for 1 hour. The
NaOH treated tissue was then put in large test tubes containing 100
ml of 0.2 M NaHCO.sub.3 buffer at pH 10.5 and 2% propylene oxide.
The tubes were gently shaken on an automatic rocker for 48 hours at
room temperature. The tissue was thoroughly washed with saline to a
pH level of 6.5-7.5, and then stored in 70% ethanol.
[0104] Preparation of GA Crosslinked Tissue
[0105] Glutaraldehyde (GA) crosslinked bovine pericardium patches
are commercially available under the tradename "Peri-Guard",
including Supple Peri-Guard.TM., and were obtained from
Bio-Vascular, Inc., St. Paul, Minn.
[0106] Sterilization
[0107] The wet tissue patches were cut into a sample size of 1
cm.times.2 cm. The samples were laid flat on a plastic wrap (four
each) and enclosed by folding the plastic wrap around. The wrapped
samples were placed inside plastic/aluminum foil pouches that were
subsequently purged with Argon gas and heat-sealed. The pouches
were sent for sterilization by electron beam radiation at 25.+-.2.5
KGy.
[0108] Implantation
[0109] The animals were 3 month old Fisher 344 male rats. Each
animal received two different material implants. Upon the surgical
procedures, the animals were anesthetized with pentobarbital (5
mg/100 g), and the upper backs were shaved and washed with a
butadiene solution. A 2 cm incision was made over the midline on
the back of the animal. The subcutaneous tissue plains were
dissected laterally to form a pouch on the left and right sides of
the back. One sample was inserted and spread flat in each pouch.
Wounds were closed with surgical sutures and washed with butadiene.
The animals were returned to their cages after recovering from
anesthesia.
[0110] Explantation
[0111] At 4 and 12 weeks post-implantation, animals were sacrificed
by carbon dioxide inhalation. The samples were retrieved together
with the surrounding adherent tissue. The retrieved samples were
cut in 3 pieces. One piece was stored in saline with 0.3% sodium
azide and used for suture retention test, the second one fixed in
Bouin fixatives and sent for embedding, sectioning and hematoxylin
and eosin ("H&E") staining, and the third piece stored frozen
and used for enzymatic digestion assays.
[0112] Suture Retention Measurement
[0113] A suture retention test that determines the force necessary
to pull a suture loop from the prosthesis was performed on the
ChemDyne MC1000 (Columbia Labs, Inc.) tensile testing system. A 5-0
Prolene suture was looped through the tissue with a 2 mm bite below
the edge of the tissue. The suture loop was pulled at a rate of 100
mm/min with sampling rate of 20 Hz.
[0114] Enymatic Digestion Assays
[0115] The tissue samples were immersed in 1.0 ml of 40 U/ml
collagenase (Worthington, Biochem Corp.) and 1.0 ml of 0.05%
trypsin/EDTA solution, respectively. The samples were incubated for
12 hours at 37.degree. C. and scored visually for tissue
integrity.
Results
[0116] Suture Retention
[0117] While GA crosslinked bovine pericardium substantially
maintained its suture retention property throughout the
implantation period (up to 12 weeks after E-beam sterilization),
there were substantial changes in the PO capped tissue following
E-beam sterilization as well as implantation (Table 7). It appears
that E-beam radiation reduced the suture retention of PO capped
tissue by about 60%. The suture retention was further reduced
during the implantation period. However, it is interesting that the
suture retention of PO capped tissue appeared to increase with time
after reaching the lowest level at 4 weeks post-implantation.
7TABLE 7 Suture Retention Force (g) of Tissue Samples Before and
After E-Beam Sterilization and Implantation in Rats Tissue
Materials GA Crosslinked (SPG) PO Capped Prior to E-Beam & 1180
.+-. 50 (n = 4) 802 .+-. 212 (n = 4) Implantation Post E-Beam,
Prior to Not Tested 300 .+-. 19 (n = 4) Implantation 4 Weeks Post
Implantation 914 .+-. 173 (n = 4) 82 .+-. 29 (n = 4) 12 Weeks Post
Implantation 1039 .+-. 145 (n = 4) 157 .+-. 39 (n = 4)
[0118] Enzymatic Digestion
[0119] GA crosslinking rendered bovine pericardium resistant to
collagenase and/or trypsin before and after implantation (up to 12
weeks). However, the PO capped bovine pericardium was readily
digested by collagenase as well as trypsin prior to implantation.
Since bovine pericardium is largely made of collagen that in its
natural state (i.e., non-crosslinked) can be digested by
collagenase but not trypsin, it is interesting that the tissue
became susceptible to trypsin after PO capping. Following
implantation, the PO capped samples were totally digestible by
trypsin at 4 weeks, but partially at 12 weeks. It is possible that
new collagen formed in the samples at later stages of
implantation.
[0120] Histological Evaluation
[0121] The histological slides (H&E stain) were evaluated under
an optical microscope and scored under a scale of 1 to 4 (Table 7).
At 4 weeks post-implantation, the GA crosslinked samples induced a
slight to moderate level of inflammatory response as characterized
by considerable amounts of polymorphonuclear leukocytes (PMN's),
macrophages, and foreign body giant cells, as well as lymphocytes,
found mainly at the outer surfaces of the implant. In comparison,
very mild or no reaction was found for the PO capped samples that
looked clean with very few inflammatory cells present. Fibrous
encapsulation was evident around the GA crosslinked tissue
implants, but almost not detectable in PO capped samples. Collagen
fiber structure in the GA crosslinked tissue matrix was unchanged,
while the tissue matrix of PO capped implants appeared delaminated
and loose.
[0122] At 12 weeks post-implantation, while the inflammatory
response to the GA crosslinked tissue was similar to that at 4
weeks with little change in the physical integrity, there were
marked changes in the PO treated samples. There were more cellular
infiltrates (especially fibroblasts) around, as well as within, the
PO treated tissue. The PO capped tissue matrix became uniform and
anisotropic with no wavy fibrous structure as observed in regular
bovine pericardium. In some regions under a thin fibrous capsule,
the tissue matrix resembled the characteristics of developing
granulation tissue with fibroblasts, neo-collagen and
macrophages.
8TABLE 7 Microscopic Evaluation (Scale of 1 to 4) of Explants at 4
and 12 Weeks GA GA PO PO Crosslinked Crosslinked Capped Capped
Parameters (4 Weeks) (12 Weeks) (4 Weeks) (12 Weeks) Poly- 2 2 1 1
morphoneuclear (PMNs) Lymphocytes 2 2 1 1 Plasma Cells 0 0 0 0
Macrophages 1 2 0 1 Giant Cells 2 2 0 1 Necrosis 0 0 0 0
Fibroplasia 0 0 0 0 Fibrosis 2 3 1 2 Fatty Infiltrate 0 0 0 0
Fibroblast 0 0 1 2 Proliferation
[0123] In conclusion, compared with the GA crosslinked bovine
pericardium, the PO capped, non-crosslinked bovine pericardium
induced less inflammation as indicated by fewer inflammatory cells
(such as PMNs and macrophages) present at 4 and 12 weeks
post-implantation. While the GA crosslinked tissue maintained most
of its physical and structural integrity throughout the
implantation period, the PO capped tissue appeared to undergo
significant changes during implantation. Following implantation,
the PO capped tissue was partially degraded within the first few
weeks resulting in decreases in suture retention. Interestingly,
however, instead of being totally adsorbed in the body the material
appeared to be remodeled over time with new host tissue and became
stronger with increasing suture retention. New collagen formation
probably occurred in the remodeling process as indicated by
fibroblast proliferation and increased resistance of the explants
to trypsin digestion at 12 weeks. Histological examination revealed
that at later stages (e.g. 12 weeks) of implantation the matrix of
PO capped bovine pericardium began to resemble the granulation
tissue, which is the specialized type of tissue that is indicative
of a normal healing process.
* * * * *