Note: Descriptions are shown in the official language in which they were submitted.
2169381
ENHANCED CROSSLINKING OF NATURAL TISSUES
TECHNICAL FIELD OF THE INVE.'~1TION
The invention concerns the use of a crosslinking
agent, such as glutaraldehyde, to process a biological
tissue, such as a heart valve.
BACKGROUND OF THE INVENTION
The preparation of bioprosthetic tissue prior to
implantation typically includes treatment to stabilize it
against subsequent in vivo enzymatic degradation.
Typically this treatment includes crosslinking molecules,
particularly collagen, on and/or in the tissue. Various
aldehydes have been used for this purpose, including
glyoxal, formaldehyde, and glutaraldehyde.
Glutaraldehyde, however, is usually the agent of choice,
in part because it may be used at physiologic pH under
aqueous conditions. In addition to crosslinking the
tissue, glutaraldehyde is a good sterilizing agent, and
provides for reducing the antigenicity of the tissue
after implantation.
Furthermore, the use of glutaraldehyde has shown
to be beneficial in producing tissues of greater thermal
' stability, greater flexibility (in comparison to
conventional crosslinking techniques), and increased
durability.
However, glutaraldehyde is also cytotoxic -- even
low concentrations of glutaraldehyde require rinsing the
tissue to remove residual glutaraldehyde. At the
concentrations typically used to crosslink biological
tissues -- about 0.6°s v/v, even though as little as 0.2%
has been used successfully -- glutaraldehyde's toxicity
is even more of a problem.
CA 02169381 1999-07-28
2
SUMMARY OF THE INVENTION
The present invention provides an improved method
for crosslinking or fixing collagenous biological tissue,
such as a porcine heart valve, using a low concentration
of crosslinking agent. The residual levels of
crosslinking agent are reduced, while maintaining or
enhancing durability and flexibility of the treated
tissue .
In one broad aspect, then, the present invention
relates to a method for treating a biological tissue in
vitro comprising: exposing a biological tissue to less
than 0.2o by volume crosslinking solution; and exposing
the crosslinked biological tissue to an anticalcification
solution selected from the group consisting of at least
one aluminum salt, a solution containing at least 500
ethanol, or combinations thereof.
In another broad aspect, the present invention
relates to a stabilized biological tissue comprising an
excised biological tissue which has been exposed to less
than about 0.2% by weight crosslinking solution, and
having a shrink temperature from about 80C to about 90C.
Since the process utilizes an aqueous delivery
system, it avoids the use of organic solvents such as
acetone, which are not only potentially toxic, but also
dehydrate, denature, or destroy collagen-containing
substrates (i.e., veins, arteries, heart valves, etc.).
Other attributes of low-concentration glutaraldehyde
fixation include: 1) a relatively higher degree of
intramolecular cross-links provides softer tissue. For
heart valves, this means improved hemodynamics; for
vascular grafts, this means greater kink resistance. 2)
A relatively higher degree of short chain intramolecular
cross-links (along the collagen strand), which may also
provide the tissue with protection from sources known to
cause chain scission reactions in the collagen protein
matrix. This is in contrast to long chain intermolecular
CA 02169381 1999-07-28
2 (a)
cross-links (between collagen strands) which are
prevalent in high concentration glutaraldehyde cross-
linking. 3) Resistance to calcification. Calcification
of bioprosthetic products, such a bio-prosthetic heart
valves, is a very complex process with a large number of
contributing factors. One of these is that calcification
tends to develop in the presence of residual free
glutaraldehyde. Fixation in low concentrations of
glutaraldehyde typically diminishes the amount of
residual glutaraldehyde in the tissue. Furthermore,
while calcification also tends to manifest itself in the
vicinity of tissue fractures, softer tissue resulting
2169381
3
from low-concentration crosslinking is less likely to
develop fractures or cracks over time.
While the present invention is suitable for a
number of vascular prostheses, it is of particular value
in the field of small diameter vessel replacement. It is
useful in coronary access bypass procedures, particularly
when the patient has had previous coronary replacement
surgery and adequate saphenous veins or internal mammary
arteries no longer exist due to previous excision. Since
the prosthesis made in accordance with *~~his invention may
have patency equivalent to or better than that of a
i
saphenous graft, the use.of such a prosthesis would
alleviate the need for saphenous vein excision. It is
also applicable to systemic microvascular vessel
replacement, such as in the hand or foot.
BRIEF DESCRIPTION OF THH FIGURES
Figure 1 compares tensile strength values of
bovine carotid arteries crosslinked in O.Olo
glutaraldehyde vs. 0.05% glutaraldehyde.
Figure 2 compares suture retention values of
bovine carotid arteries crosslinked in 0.01%
glutaraldehyde vs. 0.05% glutaraldehyde.
Figure 3 shows the effect of different fixation
conditions on tensile and suture retention strengths of
bovine median arteries.
Figure 4 shows the effect of different fixation
conditions on bursting strength of bovine median
arteries.
Figure 5 illustrates the difference between low
concentration glutaraldehyde intramolecular crosslinks
and high concentration glutaraldehyde intermolecular
crosslinks.
Figure 6 compares the leaflet shrink temperature
of leaflets crosslinked in 0.5% glutaraldehyde vs 0.05%
glutaraldehyde.
2169381
4
Figure 7 shows the effects of e-beam radiation on
pressure drop.
Figure 8 shows the effects of e-beam on effective
orif ice area .
Figure 9 shows shrink temperature versus fixation
time for aortic root tissue.
DETAILED DESCRIPTION OF THE INVENTION
A method in accordance with the invention includes
treating or processing biological tissue by exposing the
biological tissue to less than about 0.1% by volume-.
cxosslinking solution, preferably between about 0.01% to
about 0.099% by volume fixing solution. In a preferred
embodiment of the invention, the fixing solution is a
buffered solution containing glutaraldehyde.
The team "biological tissue" as used herein refers
to a collagen-containing material which may be derived
from different animal species, typically mammalian. The -
biological tissue is typically a soft tissue suitable for
implantation, such as biooros~hetic tissue or the like,
20. but the invention should not be limited thereby.
Specific examples include, but are not limited to, heart
valves, particularly porcine heart valves; aortic roots,
walls, and/or leaflets; pericardium, preferably bovine
pericardium or the like, and products derived from
pericardium, such as a pericardial patch; connective
tissue derived materials such as dura mater; homvgraft.
tissues, such as aortic homografts and saphenous bypass
grafts; tendons, ligaments, skin patches; blood vessels,
particularly bovine arteries and veins, and human
umbilical tissue, such as veins; bone; and the like. Any
other biologically-derived materials which are known, or
become known, as being suitable for processing in
accordance with the invention are within the
contemplation of the invention.
2? 69381
In accordance with the invention, the biological
tissue, explanted from its source, may be processed in
any suitable manner prior to exposure to a crosslinking
agent. Typically, the biological tissue is carefully
5 cleaned and then treated with a filtered proteolytic
enzyme concentrate to digest and thereby substantially
eliminate antigenic tissue from the biological tissue.
When natural tissue is utilized, the cleaning step
typically involves stripping the adventitia and
undesirable fat and muscle tissue from the tissue.
Exposing the biological tissue to a crosslinking
reagent, as used herein,~refers to any method of
contacting the biological tissue with the crosslinking
agent. In a preferred embodiment of the invention,
exposing the tissue to a crosslinking agent refers to
immersing the tissue in the crosslinking agent.
When tissue is immersed in a solution of a given
concentration of a crosslinking agent such as an
aldehyde, e.g., glutaraldehyde, the concentration of
glutaraldehyde within the interstices of the tissue
equilibrates with the surrounding solution so hat the
tissue experiences true low-concentration crosslinking.
However, when glutaraldehyde is delivered via a
nebulizer, monomeric glutaraldehyde is deposited directly
to the surface of the substrate. Regardless of the
initial concentration of the solution prior to
vaporization, pure glutaraldehyde is in contact with the
tissue. The concentration in the tissue is therefore
very difficult, if not impossible, to control.
The glutaraldehyde that is typically used in
accordance with the invention is a biological grade 50°s
solution commercially available, from, for example,
Electron Microscopy Sciences (Fort Washington, PA). Such
commercially available glutaraldehyde may also be
available in a variety of other grades, purities, and/or
concentrations. A biological grade of glutaraldehyde
~~~938~
6
typically does not require additional purification, but
it may be desirable to pass the final crosslinking
solution through a 0.2u pore size hydrophilic membrane
filter to remove any biological contaminants.
In accordance with the invention, a biological
tissue may be exposed to a fixing solution comprising
glutaraldehyde in a suitable buffer. Suitable buffers
for use in the practice of the invention are those
buffers which have a buffering capacity sufficient to
maintain a physiologically acceptable pH, do not cause
substantial deleterious harm to the biological tissue,
and/or do not interfere~with the treatment process.
Exemplary buffers include, but are not limited to
phosphate-buffered saline (PBS), and organic buffers,
such as N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic
acid) (HEPES) or morpholine propanesulphonic acid (MOPS);
and buffers which include borate, bicarbonate, carbonate,
cacodylate, or citrate. The preferred fixing solution is
less than 0.1% v/v glutaraldehyde in.a citrate buffer.
In a typical protocol according to the invention,
the biological tissue maybe exposed to the fixing
solution for a time and at a temperature sufficient to
induce crosslinking of the collagen in and on the
biological tissue. For example, the biological tissue
may be exposed to a buffered glutaraldehyde solution from
about 4C to about 37C, preferably at about 20C; at a
pH from about 6 to about 8, preferably about 6.3 to about
6.5; and for a period up to about 10 days, preferably
from about 2 to about 5 days.
In accordance with the invention, one skilled in
the art will recognize that certain parameters in the
treatment protocol may be varied according to achieve a
particular purpose. These parameters include, but are
not limited to glutaraldehyde concentration, solution
composition, pH and ionic strength, time and temperature
of biological tissue exposure to glutaraldehyde, and the
2169381
ratio of tissue to volume of solution, and the biological
tissue configuration during the initial fixation. The
invention is not to be limited thereby.
Typically, the crosslinked biological tissue is
then rinsed, using, for example,-any suitable rinsing or
laving material. In a preferred embodiment, the rinsing
agent is sterile, physiological saline.
The tissue may be rinsed with many volumes.of
sterile, physiological saline over a period of
approximately 24 hours, or until the concentration of
residual processing chemicals in the tissue are below
levels Which are considered to be toxic (approximately 1
PPm) -
One skilled in the art will also recognize that
the invention may include additional processing. For
example, the crosslinked biological tissue may be exposed
to one or more anticalcification agents, one or more
bioburden reduction agents, at least one rinsing -
solution, and one or more sterilizing agents or
protocols. Furthermore, as shown in more detail below,
the biological tissue, cr~sslinked in accordance with the
invention, may be stored for up to about one year or more
prior to final sterilization. Exemplary additional
processing is described in more detail below.
BIOBURDEN REDUCTION
An embodiment of the invention may include
exposing the crosslinked biological tissue to one or more
bioburden reduction agents, typically for up to about 10
hours, preferably for about 2 to about 4 hours. For
example, a porcine heart valve treated with
glutaraldehyde as noted above may then be exposed to a
buffered solution containing about 1-5% glutaraldehyde,
about 1-6% formaldehyde, and about 15-25% ethanol.
Typical buffers include PBS, KEPES, phosphate, and
citrate buffers.
2? 698?
INTERMEDIATE STORAGE
An embodiment of the invention may include storing
the crosslinked biological tissue for up to a year or
more prior to final sterilization. For example, a
porcine heart valve crosslinked with glutaraldehyde in.
accordance with the invention may be temporarily stored
at room temperature in a 0.5% glutaraldehyde solution
buffered with PBS, citrate or the-like.
ANTICAhCIFICATION TREATMENT
An embodiment of the invention may include
r _
exposing the crosslinked tissue to one or more reagents
designed to reduce or inhibit calcifica~ion of the
biological tissue after implantation. A number of anti-
calcification reagents are known in the apt. For
example, the crosslinked biological tissue may be exposed
to an alcohol and/or an aluminum salt in order to reduce
or inhibit calcification. In an exemplary process, the
crosslinked biological tissue may be immersed in a
solution containing greater than about 50% of a lower
aliphatic alcohol for a period sufficient to render the
biological tissue resistant to calc~fica~ion, typically
up to about 96 hours.
The alcohol is preferably a lower aliphatic
alcohol (C1 to C3), such as methanol, ethanol, propanol
or isopropanol. In a preferred embodiment, the alcohol
is ethanol.
The length of time for exposure to the alcohol
treatment can be varied by those of skill, in the art.
For embodiments of the invention wherein the biological
tissue is immersed, or soaked, in a liquid treatment
solution of the alcohol, an illustrative exposure time is
preferably between about 24 to 96 hours.
In another embodiment of the invention; the
anticalcification agent may include a multivalent
metallic cation, such as a salt of aluminum or iron. For
2169381
9
example, the crosslinked biological tissue may be
immersed in a solution containing from about O.1M to
about O.OO1M A1C13 for a period sufficient to render the
biological tissue resistant to calcification.
In accordance with the invention, the crosslinked
biological tissue may be stored in an alcohol-
glutaraldehyde solution, preferably in an amount
sufficient to maintain calcification inhibition and/or
sterility. For example, the biological.tissue may be
stored in a buffered alcohol solution containing
glutaraldehyde, typically greater than about 60%, and
preferably between about~60% and about 80%, alcohol and
less than about 0.5%, preferably between about 0.2% to
0.5%, glutaraldehyde.
The biological tissue, calcification-inhibited
and/or crosslinked, may then be placed or packaged in a
container. In accordance with a preferred embodiment of
the invention, the biological tissue is packaged and
sealed, in physiological saline, in its final container
prior to terminal sterilization. Packaging preferably
means placing in a container suitable for sterilization,
storage, and/or shipping.
The container may be constructed of glass or
polymeric plastic, such as polypropylene, polyethylene,
and/or epoxies. It is intended that the invention should
not be limited by the type of container and seal being .
employed; other materials may be used, as well as
mixtures, blends, and/or copolymers.
The crosslinked, packaged biological tissue may
then be sterilized, as noted below, or it may be stored
for up to about a year or more prior to sterilization.
In accordance with the invention, storage includes long
term storage, e.g., six months, twelve months, or for up
to about five years or more.
STERILIZATION
21b9381
A method in accordance with the invention may also
include sterilizing the tissue. The term "sterilizing"
as used herein refers to exposing the biological tissue
to a sterilizing beam of accelerated electrons, i.e., an
5 electron beam (e-beam). The particle beam which
comprises the-e-beam preferably includes directional
bombardment, i.e., bombardment from one direction only,
and includes single-side or multiple-side irradiation.
The biological tissue, crosslinked in accordance with the
10 invention, may be sterilized, preferably after the
i
biological tissue has been packaged. Suitable
sterilizing protocols include, but are not limited to x-
ray or gamma radiation, e-beam radiation, and the Like.
The preferred method of sterilizing the crosslinked
tissue is by exposing the biological tissue, packaged in
saline, to accelerated electrons. For example, the
biological tissue may be subjected to the electron beam
until a dose of approximately 25 kilogray (kGy) is
achieved, or approximately 1-10 minutes, depending on the
dimensions of the material.
A major advantage of e-beam processing over
conventional gamma radiation is the processing speed or
the high rate at which the energy can be applied in a
controlled manner, which usually translates to lower
sterilization costs.
In accordance with the present invention,
biological tissues are treated by exposing the tissue to
e-beam radiation sufficient to effect sterilization.
Additionally, the present invention provides a biological
tissue sterilized by e-beam radiation, with the resulting
biological tissue exhibiting enhanced performance
characteristics. The methods and tissues according to
the present invention have the added advantage of reduced
risk of infectivity, and eliminates the need for aseptic
handling protocols. Further, the methods and tissues of
the present invention, which use fewer reagents and/or
2169381
11
require less processing, provide for lower costs in
labor, reagents, time and personnel.
E-beam radiation sterilization is effective in
obviating the need for toxic sterilizing chemicals.
Moreover, the amount of radiation required for e-beam
sterilization does not significantly degrade the
biological tissue, thus providing a more durable
transplantable tissue.
The dose rate for gamma radiation is approximately
110 grays per minute and the dose rate of e-beam is
approximately 7800 grays per minute. Consequently,
exposure times are dramatically greater for gamma
radiation, which requires low doses over an extended
period to effect sterilization. In contradistinction to
gamma radiation, the high dose rates involved in e-beam
irradiation promote diffusion of oxygen into biological
tissue at a rate insufficient to participate in free
radical formation reactions, such as those which might
contribute to tissue and polymer degradation. This is
particularly advantageous in those embcdiments which
include placing the biological tissue in a container
prior to irradiation, since polymer degradation in both
the tissue and the container may be minimized.
Furthermore, the high dose rate of e-beams
relative to gamma rays permits a higher processing rate
of sterilization, commonly an order of magnitude higher.
In relative terms, gamma radiation penetrates
approximately ten times further into materials than 10
MeV electrons in the same material.
Gamma rays induce excitation of electrons within
the atoms of the materials to be sterilized. Electron
beams, on the other hand, provide high-energy electrons
to the exterior of the material, which penetrate the
material, and in turn put subsequent electrons in motion.
z ~ ~~3s ~
12
Because of the relatively high dose rate of the
e-beams, oxygen is not capable of diffusing into the
material at a rate required to participate in oxidative
reactions that may lead to degradation of the material.
Furthermore, prevention of degradation in both the
package and the tissue permit terminal sterilization,
i.e., sterilization of the tissue in its final, sealed
package. Thus, the present invention avoids the need for
costly aseptic handling techniques, and provides
l0 sterility assurance as Long as the package is intact,
i.e., until the tissue is ready for use.
As high energy electrons penetrate the surface
they callide with atomic electrons of the material.
recoil and collide to set more
in turn
These electrons
,
,
electrons in motion so that frcm a relatively few
electrons penetrating the sur~ace, there results a
multiplicity of electrons depositing energy in the
primarily by the production of ions and free
material
,
radicals. This process, called buildup, results in
higher doses being delivered to depths below the surface
where the primary beam and its recoil electrons can no
longer produce ionization.
' In accordance with the invention, the amount of
e-beam radiation is an amount sufficient to sterilize the
biological tissue, and in some embodiments, an amount
sufficient to sterilize the biological tissue packaged in
its final container. One skilled in the art will
recognize and be able to determine a sterilizing dose and
time suitable far a particular tissue and based on the
characteristics of the accelerator being used.
Typically, the biological tissue is subjected to a
one-sided exposure to the electron beam until a
sterilizing dose of radiation is absorbed. Absorbed dose
of radiation is expressed in terms of kilog:ays (Kgy).
One kilogray is equal to one thousand joules of energy
deposited per kilogram of material. For example, the
13
biological tissue may be irradiated until a dose of
_ approximately 25 Kgy or more is achieved.
Effective sterilization may be easily determined
using conventional microbiological techniaues, such as
for example, the inclusion of suitable biological
indicators in the radiation batch or contacting the
tissue with a culture medium and incubating the medium to
determine sterility of the tissue. Dose may also be
determined with the use of radiochromic dye films. Such
films are calibrated, usually in a ga.~una field, by
reference to a national standard.
Degradation of the biological tissue by
irradiation may also be determined using well known and
conventional tests and criteria, e.g. reduction in shrink
temperature, T,; susceptibility to enzr.~e attack, e.g.
collagenase; extractability of degradation products, e.g.
collagen fragments; and decrease in physical properties
such as tensile~strength.
In a typical protocol according to the invention,
the biological tissue may be exposed to the fixing
solution for a time and at a temperature sufficient to
induce crosslinking of the collagen in and on the
biological tissue. For example, the biological tissue may
be exposed to a buffered glutaraldehyde solution from
about 4°C to about 37°C, preferably at about 20°C; at a
pIi from about 6 to about 8, preferably 6.3 to 6.5; and
for a period up to about l0 days, preferably from about 2
to about 5 days.
The aforementioned steps, in combination, produce
a prosthesis having greater strength and pliability,
reduced antigenicity, and greater ease of use than
prostheses produced using other processes.
LONG TERM STORAGE
In another embodiment of~the invention, the
biological tissue, crosslinked in accordance with the
21b~381
14
invention, may be packaged in a solution of approximately
0.5% glutaraldehyde. Using aseptic techniques, the
biological tissue may be thoroughly rinsed with sterile,
physiological saline (e.g., 0.9% sodium chloride). The
purpose of the saline rinse is to reduce the
concentration of residual glutaraldehyde present on the
tissue surface and in the interstitial spaces of the
tissue, thus minimizing the chance of a toxic response in
the patient. The biological tissue may be placed into a
plastic or polymeric container, filled with sterile
~ saline, and capped or. sealed. The capping of the product
should be a permanent-seal that should not be opened
until the time of the implant.
The present invention provides an improved
biological tissue in view of its strength, pliability,
and reduced antigenicity. These characteristics are
particularly desirable in view of the stressful procedure
required to implant the biological tissue and the-
possibility of rejection by the body. With respect to
the implantation procedure for some biological tissues,
the biological tissue must have resistance to kinking,
good suture puncturability, and the ability to readily
seal suture holes. Suture retention, i.e., the ability
to resist tensile force, must be high. Additionally, the
.. 25 graft must have high resistance to bursting to withstand
possible high systolic pressures and to guard against
. aneurysm fortnativn. Grafts that are insufficiently
strong and/or are too antigenic may be potentially fatal
to the patient.
The prostheses produced according to the present
invention may be packaged as a kit, preferably sterile
wet-packaged in a 0.9% NaCl solution. The selection of
such additional elements are well within the ordinary
skill in the art.
EXAMPLES
z ~ 693s ~
Examgle 1. Fresh tissue (e. g., blood vessels, hearts,
heart valves, or pericardium) are procured from a local
processing facility (bovine, porcine, ovine, etc.) and
received in physiological saline (0.9~ sodium chloride)
5 on ice. The tissue is either dissected immediately or
placed in fresh sterile saline and refrigerat8d
overnight. Extraneous tissue such as adipose, skeletal
muscle, myocardium, bane, trachea, etc., is carefully
removed from the tissue of interest. The tissue is then
10 again washed and immersed in fresh sterile saline.
~ Although-this technology works to varying degrees
at a range of glutaraldehyde concentrations,
approximately 0.03% v/v provides radioprotective
properties and the crosslin:c~ng time fits reasonably well
15 within a manufacturing schedule. For 10.0 liters of 50
mM_ citrate buffered 0.03% (v/v) glutaraldehyde:
Step 1 )-
A 50 mM_ citrate buffer solution is prepared
per the following formula (l0 liters):
To 9.0 liters of sterile, de-ionized water,
add:
140.0 grams of Sodium Citrate
5.0 grams of Citric Acid Monobasic
.. 38.6 grams of Sodium Chloride
Bring the volume of the solution up to 10.0
liters with sterile, de-ionized water
Step 2)
To 9.0 liters of the 50mM citrate buffer
solution prepared in Step 1, add 6.0
milliliters of 50% Biological Grade
Glutaraldehyde
Bring the solution volume up to 10.0 liters
using the SOmM citrate buffer solution
prepared in Step 1.
216938
t6
step 3)
Adjust the pH vt the soluti~rr to 6.40 ~ 0.0~
wing hydrochloric acid or sodium hydroxide_
The tissuQ is then immArsed in the glutaraldehyde,
~ solution, at room temperature (20-25°C) for the
crosslinking reactlnn_ As fixation time progresses,-the
_ n~,er of crosslinks increases, ao shown in thQ form of a
shrink temperature curve (See Figure 9). The
concentration of glut~raldehyde in solution decreaePC as
to it is consumed by the tissue in the tuzm of poly-
glutaraldchydc crvsslinks. ThQrefore, .it may be
desirable to replenish the tixd~i.un solution at interval~-
throughout the crosslinking roactinn. Because a majority
of the crosslinks are foz-med early, it is rccommcnded to
change thQ solution approximately eight hours Lollowinq
the onset of the reaction, then daily thereafter.
The exposure o~ t.issue to the glut8raldehydE
solution procced~ for a period of time -ranging from about
24 to about 120 hours, depending on the concentration of
glutaraldehyde in the solution. In general, a high
glutaraldehyde concentration corresponds to a short
fixation time; a loW glutaraldehydp concentration
corresponds to d long Fixation tim . For a 0.03$
solution, an exposure time of approximately 72 hours is
sufficient to maximia~ the crosslink density within the
interstices of the tissue. .'this corresponds to a shrink
temperature of approximately so-89°C, depending on the
type of tissue used.
When the crosslinking reaction has ended, the
tissue is submersed in a sterilant solution containing 2$
(v/v) glutaraldehyde, 3% (v/v) formaldehyde, and 20~s
(v/v) ethyl alcohol. This multi-component sterilant
reduces any residual bioburden on the tissue prior tv
rinsing and packaging. .
21 b9381
17
The tissue is then thoroughly rinsed with
sufficient sterile saline to minimize the presence of the
processing chemicals. This typically requires applying
four or five 10 liter aliquots over a 24-hour period.
The exposure time should be watched carefully, since
diffusion of residuals from the tissue is a time-
dependent phenomenon. After the final rinse, t:~e tissue
is placed in a sterile container (valve_jar, vascular
graft vial, etc.) and filled with sterile saline. The
package is then permanently sealed. Note: all
~ manipulations of the tissue subsequent to the bioburden
reduction process with the multi-compcnent sterilan~
should be performed as aseptically as possible to
minimize the extent of contamination crior to e-beam
sterilization.
Example 2. Two identical experiments were performed to
evaluate the effects of different concentrations of
glutaraldehyde on the physical properties of bovine
carotid artery vascular protheses. ~rteries were
received in cold, sterile saline (0.9% sodium c:~loride).
Extraneous tissue such as adipose, bone, cartilage,
connective tissue, etc., was stripped from the vascular
tissue. The arteries were enzymatically digested with a
citrate-buffered ficin solution to remove a specified
portion of smooth muscle tissue. The arteries were
thoroughly rinsed and placed into one of tao tanks
containing glutaraldehyde to crosslink the collagen
component of the tissue. One tank contained 50mM
citrate-buffered 0.01% glutaraldehyde, the other tank
contained 50mI~ citrate-buffered 0.05% glutaraldehyde.
The arteries in the 0.01% solution were allowed to
crosslink for approximately five days and the arteries in
the 0.05% solution were crosslinked for approximately two
days. Upon completion of the crosslinking reaction, all
2169381
is
arteries were placed in a 50mM_ citrate-buffered 2%
glutaraldehyde solution as a sterilization step.
Tissue samples were then submitted for evaluation
of Radial Tensile Strength and Suture Retention Strength.
The Radial Tensile Strength test involves cutting a piece
of crosslinked carotid artery, making a longitudinal
incision, and pulling the tissue in a radial orientation
until failure on an apparatus such as an Instron. The
Suture Retention Strength test involves inserting a loop
l0 of surgical suture, such as a 5-0 PTFE-impregnated
y polyester suture, through a tissue sample a specified
distance from a cross-sectionally cut edge. This
distance, the bite size, may be, for example, about 3 mm.
The suture is pulled until the tssue fails on an
apparatus such as an Instron. The results of the Tensile
Strength and Suture Retention Strength testing is
displayed on Figure 1 and 2, respectively.
The results of the Tensile Strength testing show
there are no significant differences in the strength of
the tissue that can be attributed to variation in
fixation solution concentration. The seemingly wide
error bars are due to the inherent diversities found in
biological tissues.
_ There appears to be a trend in the Suture-
Retention Strength, specifically, strength for both
concentrations is lower in Batch 2 and Batch 1. This is
most likely a result of a difference in the enzymatic
digestion processes. Each ficin solution varies slightly
in enzymatic activity. In other words, it is possible
that the digestion solution prepared fvr Batch 2 had a
slightly higher enzyme activity than that for Batch 1.
Nevertheless, the differences in strengths within the
batch are negligible.
Examine 3. An experiment was performed to evaluate the
effects of different concentrations of glutaraldehyde on
~i6938~
19
same of the physical properties of bovine median artery
vascular protheses. Processing of tissue was the same as
that described in Example 2 until the crosslinking step.
The digested arteries were placed in one of three tanks
for crosslinking. Tank 1 contained 50mM citrate-buffered
0.01% glutaraldehyde, tank 2 contained 50mM_ citrate-
buffered 0.075%.glutaraldehyde, and tank 3 contained 50mM_
citrate-buffered 2.0% glutaraldehyde. The arteries were
allowed to remain in each of the fixation tanks until the
crosslinking reaction was completed, or about four days.
Upon completion of the crosslinking reaction, half the
arteries from tanks 1 and 2 were placed in a 50mM_
citrate-buffered 2% glutaraldehyde solution as a
sterilization step. The other half re:~ained in the
original solution. After 24 hcurs, all arteries were
placed in individual glass vials containing 40~ ethyl
alcohol, and capped. Samples from each group were then
subjected to the following test: Radial Tensile
Strength, Suture Retention Strength, and Bursting
Strength.
Figure 3 shows that the radial tensile strength
and suture retention strength are very similar for tissue
processed at each of the conditions in this experiment.
The extremes (0.01% and 2.0%) are of particular interest
because this data represents the lower end of the range.
This data contradicts the very common notion in the
industry that maintains that high concentration fixation
leads to high thermal stability, which leads to high
strength. These results, as well as the shrink
temperature data in Figure 6 show that this is not the
case. The fact that the 0.01%/2% group has a sightly
lower tensile strength than the other groups is
considered an artifact. It is expected that the
subsequent treatment with 2% glutaraldehyde will, if
anything, strengthen the tissue, not weaken it.
21b938~
Figure 4 shows the results of the Bursting
Strength testing, where a vascular prosthesis sample is
inflated with water until it bursts. The internal
pressure at the time of failure is recorded as the
5 bursting strength. Again, looking at the ex~~emes,
results are nearly identical, even though the low-
glutaraldehyde test group was fixed at a glutaraldehyde
concentration 1/2ooth of conventional 2% crosslinking.
Example 4. Figure 5 is a simplified representation of
10 the formation of a short-chain intramolecular crosslinks
produced by low-glutaraldehyde and long-chain
internolecular crosslinks produced by high-glutaraldehyde
fixation. This diagram aids in shoT~ing that, in the
presence of intramolecular crosslinks, the collagen
15 strand may maintain much of its integrity, even if
peptide bonds are cleaved by radiation exposure.
It is possible to analyze the crosslink density of
tissue by measuring the shrink temperature, or
denaturation temperature, of a given sample. Tissue
20 crosslinked with low glutaraldehyde forms'a higher '
density of intramolecular crosslinks, and this may be
expressed in terms of a higher shrink temperature than
conventionally-fixed tissue. This relationship is shown
by crosslinking tTao groups of fifteen porcine aortic
valve leaflets with 0.5% or 0.05% glutaraldehyde. Shrink
temperatures were measured via Differential Scanning
Calorimetry and the results are contained in Figure 6.
Example 5. Experiments have shown that
glutaraldehyde-crosslinked tissue, exposed to e-beam
radiation, exhibits enhanced hemodynamic performance
characteristics, such as flexibility. Evidence of
increased flexibility is provided by measuring pressure
drop across the heart valve (the change in pressure from
the inflow side of the valve to the outflow side), as
2169381
Z1
shown in Figure 7. Enhanced flexibility is also shown by
measuring the effective orifice area, the cross sectional
area through which blood flows, as srown in Figure 8.
These tests show that exposing heart valves to e-beam
radiation results in softer leaflets which tend to open
more readily and to a greater extent than non-irradiated
valves. This provides both short-term and long-term
benefits to the recipient because a larger effective
orifice area results in greater cardiac output and
IO therefore, an increase in efLiciency of cardiac activity
y and a decreased tendency to develop cuspal fractures
leading to eventual calcification and valve failure.
Eight heart valves ~we=a glutaraldehyde crosslinked
as shown in Example 1 and exposed to e-beam radiation.
The pressure drop across the heart valve before
subjecting the heart valve to e-beam radiation was
compared to the pressure drop after subjecting the heart
valve to E-bean radiation. Figure 7 g=aphically
illustrates that the pressure drop decreases when tested
on a steady state in vitro flow tester. As a reference
point, the pressure drop for a straight, unobstructed
tube would be zero.
Figure 8 compares the effective orifice area
before and after exposing the heart valve with e-beam
radiation, and shows that the effective orifice area
increases following e-beam radiation. .
Effective Orifice Area determinations were made by
placing test valves in a Pulse Duplicator system. The
Pulse Duplicator is capable of calculating a number of
valve-related functions by measuring pressures and flow
rates at strategic locations within a simulated heart
containing the test valve.
Effective Orifice Area (EOA) is defined as
follows
EOA=Q,m,/ ( 51. 6 ~ DP) , expressed in cm2, where
21 X9381
22
root mean square flow rate obtained
during the period of positive pressure drop,
in ml/second
mean positive pressure drop, in mm Hg
The theory behind enhanced hemodynamics in
irradiated tissue heart valves involves the disruption of
_ molecular bonds which hold_the collagen triple helix
intact. The intramolecular crosslinks offered by this
technology serve as reenforcement to the collagen
backbone as its own structural frame work is weakened by
the radiation. A dose of 25 kGy, in the presence of
sufficient intramolecular crosslinks, weakens the protein
framework to sufficiently render the tissue mere
flexible, yet the tissue perforc~ance improves.
Similar results have been obtained every time
these two experiments were repeated. While the exact
mechanism is unknown, it is theorized that a scission
reaction occurs within the collagen molecule. Bonds that
hold the collagen chain together appear to be broken when
subjecting a tissue to e-beam radiation. However, the
presence of intramolecular glutaraldehyde crosslinks
. appears to keep the primary structure of the collagen
molecule intact, thus maintaining the integrity of the
softened tissue.
Example 6. The major criticism of radiation as a
sterilization method for biological tissues is its effect
on long-term durability of the product. The FDA
currently requires that tissue valves demonstrate the
ability to withstand 200 million cardiac cycles on an
accelerated wear tester. This translates to
approximately five years of real time. At some point in
the future, 380 million cycles of the same testing may be
required.
23
An experiment was performed an experiment to
determine the effects of e-beam radiation on the wear-
resistance of tissue valves. Four groups of valves were
tested:
Group 1 Crosslinked in 0.03% glutaraldehyde;
stored in 0.5% glutaraldehyde (e-beam
negative control).
Group 2 Crosslinked in o.03% glutaraldehyde;
rinsed for removal of residuals;
l0 stored in 0.9% sodium chloride;
e-beam sterilized, 25 kGy.
Group 3 Crosslinked in 0.03% glutaraldehyde;
treated with anticalcification process;
rinsed far removal of residuals;
stored in 0.~% sodium chloride;
e-beam sterilized, 25kGy.
Group 4 Crosslinked in 0.5% glutaraldehyde;
rinsed for removal of residuals;
stored in 0.9% sodium chloride;
e-beam sterilized, 25 kGy
(concentration negative control).
Results of this experiment are located in Table I
below. These results clearly indicate that, compared to
control valves (Groups 1 and 4), exposing the tissue
valves to e-beam radiation does not have a negative
effect on durability after in vitro testing at 389
million cardiac cycles. The group with the best wear
data, in fact, was the group that had been exposed to e-
beam after a treatment for anticalcification.
21b9381
24
Table 1.
Results of 389 b5llioa Cycles Aaxler sled Wear Testing:
e-beam ~. No e-beam
Treatment ~ Number of Summary of anomalies
~
ValYes
Group 1 4 6 turns holes (> lmm)
Z small hobs (< lmm)
large tears in leaflets
(2-6 mm)
1 small abrasion
i valve with no observed
wear
Group 2 4 2 lame holrs
5 small bolas
l small abr~sioa
1 valve with no observed
wear
Group 3 6 ~ 3 small holes
~ valves with no observed
wear
Group 4 3 ~ 3 holes (0.~ to 3 mm)
2 valves with no observed
wear
Example 7. A vascular graft prosthesis, for example an
artery, is stripced of its adventitia, fat and muscle
tissue; and digested in an activated protease. This
stripped and cleaned artery is treated with succinic
(, anhydride at a controlled basic pH to produce a negative
_ charge on the surface of the artery. The negatively
charged artery may then be treated with glutaraldehyde in
a concentration ranging from about 0.005 to about 5.% in a
citrate buffer to fix and strengthen the artery by cross-
linkage. The preferred concentration is less than about
.O1%. In accordance with the present invention, low
concentrations of glutaraldehyde typically yield softer
and more flexible products, based on qualitative
comparative assessments of graft products cross-linked
using low and high glutaraldehyde concentrations.
Qualitatively, increased pliability of graft products
crosslinked using low glutaraldehyde concentrations was
demonstrated by assessing the radius of curvature of the
graft; the radius is smaller in low glutaraldehyde
2169381
concentration fixed product than in a high glutaraldehyde
concentration fixed product.
The fixed graft may then be s~~rilized using
glutaraldehyde at a concentration range of about
5 1% to about 4%, preferably about 2a.
This combination of steps produces a prosthesis
having increased strength, increased durability,
increased pliability, and decreased t::rombvgenicity.
The digestion step in the present invention
10 comprises using an activated protease to remove antigenic
material from the surface of the graft. While it is
known to utilize the proteolytic enzyme ficin as part of
the cleaning step in preparing a blood vessel, t:~e
present method provides more effective elimination of
15 antigenic tissue from t::e natural graft prosthesis
through the use of an activated protease. What remains
after the digestion step is a collagen matrix.
Example a. After harvesting, the graft is digested in a
protease, preferably, an activated protease, to remove
20 antigenic substances from the graft. In a preferred
embodiment, the activated protease is ficin, more
preferably, filtered ficin that is activated by adding
cysteine to the ficin concentrate. In a preferred
embodiment, a buffer is used comprising citric acid and
25. sodium citrate, although other buffers may be used.
The specific amount of ficin used in the practice
of this invention depends on the activity of the ficin
used, since variances in the activity level of enzymes
are normal. To make up differences in the activity
levels of the ficin used, the exact amount of ficin
required to manufacture grafts is typically adjusted each
time grafts are manufactured to achieve a constant
volumetric activity; i.e., the determination of the
specific quantities of ficin to be used during graft
production typically requires that the ficin be evaluated
2169381
26
for activity and that adjust:~ents to the amount of ficin
used be made in accordance to the activity noted. The
concentration of ficin typically used according to the
present invention amounts to the addition of
approximately 65 grams ef ficin to a reactor volume of
about 20 liters. In a preferred embodiment, the ficin
activity of the digesting solution is about 9.8 mmol
NPZG/liter=minute. _
During digestion, the temperature and pH should be
monitored. In one embodiment, the temperature should be
in the range between 30°C and 50°C, more preferably 40°C
~
2°C. The pH should be in the range between 5 and 7, more
preferably 6.3 ~ 0.1. The digestion time utilized is not
critical, but should be sufficient to remove any
antigenic material; 2-3 hours is typically sufficient.
The graft should then be rinsed several times in
distilled water. In a preferred embodiment, the graft is
rinsed 4 times. -Digestion is terminated in a stop bath,
preferably a stop bath containing sodium chlorite. In a
preferred embodiment, the concentration of sodium
chlorite is 0.1~. The graft is then rinsed again,
preferably 4 times.
Example 9.
Using a hemostat, suspend one artery from a ring
stand. Using a clean scissors, strip adventitia, fat,
and muscle tissue from the artery. Place the artery in a
clean beaker with clean 0.9% sodium chloride on ice.
Repeat above steps until all arteries have been stripped.
Tie off side branches with a suture. Tie off as
close to the main artery as possible with a triple
surgeon's knot. Trim excess side branches near the knot.
Remove the artery from the ring stand When all side
branches have been tied.
Fill a large syringe with 0.9% sodium chloride
solution and attach the proper leak test fitting to the
2169381
27
luer tip. Using a hemostat, clamp one end of the artery.
Insert the tip of the syringe into the opposing end of
the artery. While securing the end of the artery to the
syringe tip with one hand, gently inject saline into the
lumen of the artery to detect leaks. Close any leaks
with a 3-0, 4-0, or 5-O suture. Tie off the suture with
a triple surgeon's knot. Continue to suture leaks in the
artery until it is leak tight under moderate pressure on
_ the syringe. Place sutured artery in a clean beaker with
clean 0.9% sodium chloride solution on ice. Repeat above
y steps until the desired number of arteries have been
sutured.
Dissolve 420 grams of sodium citrate and 14.4
grams of citric acid in H,0 and adjust the volume to 21
liters. Measure the pH and adjust to 6.3 if necessary.
The overall citrate concentration is 71.3 mM with 95.4%
sodium salt and 4.6% acid fore.
Fill the reactor with 17.32 liters of ficin buffer
and turn the mixer on. Place the heater coil in the tank
and turn the hot water on to start heating the buffer to
40C. Insert the threaded hose barb fittings into the
arteries and use cable ties to fasten them. Attach the
arteries to the graft manifold using the threaded
fittings. Attach the graft manifolds to each other and
to the graft manifold support assembly using 3/8"x2"
stainless steel pegs and place in the reactor. Start the
pumps with a flow rate of 1.05 liters/minute through each
manifold. Add the activated ficin concentrate solution
when the reactor temperature stabilizes at 40C.
The concentrated ficin solution is made to the
equivalent of 85 grams/liter of Sigma latex powder.
Place 2.8 liters of ficin buffer in a flask in the 40C
water shaker bath. Approximately 65 grams of f icin
powder are required. The ficin powder is dissolved in
the 40C buffer and filtered through a 5 micron Gelman
Acro 50A filter.
2169381
2a
Measure out 2.63 litsrs of the filtered enzyme
concentrate into a flask and place in the 40C water
bath. Weigh out 6 grams of L-cysteine and add to the
enzyme concentrate for activation. After 5 minutes, add
the enzyme concentrate to the reactor and begin
digestion. Digestion takes place for 2~ hours.
Temperatsre is maintained at 40C +/- 2C and the pH is
monitored and should remain at 6.3 +/- 0.1.
Examine the arteries periodically. Eliminate
w 10 excessive kinking to allow for even flow distribution
~~ across the manifold. .The arteries change in appearance
_ within 20 minutes from a pinkish to a silvery color. The
lumen dilates somewhat and the length of the arteries
increases about 35% during digestion. Do four full water
rinses at the end of digestion.
For the stop bath, dissolve 20 grams of sodium
chlorite into 20 liters of h=O.- Shut off the pumps and
stirrer. Open drain valve and allow the tank to empty.
Close the drain valve and fill the reactor with 20
liters of H,O. Turn on the pumps and stirrer and allow to
equilibrate for several m=notes. Repeat for a total of
four rinses.
Shut off the pumps and stirrer and open the drain
valve to allow the tank to empty. Close the drain valve
. 25 and fill the reactor with the stop bath solution. Start
the pumps and stirrer. Run the stop bath for 15-20
minutes to inactivate any residual enzyme. Drain the
reactor and perform four full rinses.
Dissolve 1680.2 grams of sodium bicarbonate in HZO
and dilute to 20 liters. Shut off the pumps and stirrer
and drain the last rinse out of the tank. Fill the
reactor with, the 20 liters of the carbonate solution.
Turn on the pumps and stirrer and allow to equilibrate
for 15 minutes.
Weigh 142.8 grams of succinic anhydride. Add i of
the anhydride to the reactor after the 15 minute
269381
29
equilibration time. Disperse the crystals into the
solution so that the stirrer can mix them throughout the
tank. Add the second half of the anhydride 30 minutes
after the first addition. The charging should take
another 60-90 minutes. Charging is complete when all of
the crystals are dissolved and bubbles are no longer
forming in the solution. When charging is done, stop the
pumps and stirrer, drain the tank and perform four full
rinses.
Measure 8.0 liters of K,O and place in a clean
carboy. Weigh out 112.0 g of sodium citrate and add to
the carboy. Weigh out 4.0 g of citric acid and add to
the carboy. Places a magnetic stir bar in the carboy.
Place the carboy on a stir plate and mix until all solids
I5 are dissolved. Citrate concentration should be 50 mM
consisting of 95.2% sodium citrate and 4.8% citric acid.
Measure 1.6 ml of 50% glutaraldehyde and add to
' the citrate buffer while it is still on the stir plate.
Allow solution to equilibrate for 10-15 minutes. Test pH
and adjust.as required to 6.~0 T/- 0.05 using 6M NaOH or
6M HC1.
Place a clean fixation tank under the exhaust
hood. Place a fixation rack into the fixation tank (2
levels for a standard.batch). Transfer 0.01%
glutaraldehyde solution from the carboy to the fixation
tank. Maintain crosslinking conditions for up to
approximately 120 hours, changing the glutaraldehyde
solution periodically, i..e., every 24 hours.
Remove the manifold from the reactor. Using a
clean scissors, cut an artery on the distal side of the
collateral ulnar bifurcation. Place the artery in a
beaker of demineralized water. Repeat above steps until
all arteries have been removed from the manifold.
Remove one graft from the beaker. Extend the
graft fully length-wise. Place the graft on a proper
size mandrel and extend it~fully length-wise. Place the
2i693gi
mandrel with the graft into a spot on the fixation rack
in the fixation tank. Repeat above steps until all
grafts have been put on mandrels and placed in the
fixation tank. Note time into the fixation tank and
5 cover the tank.
When the grafts have in been the fixation solution
for 24 hours, prepare a fresh batch of 0.01%
glutaraldehyde and transfer the solution to a clean
fixation tank. Transfer the entire fixation rac!c
l0 (including grafts) from the existing fixation tank to the
~~ fresh glutaraldehyde solution. Discard the old
glutaraldehyde. Place the tank with the fresh
glutaraldehyde under the exhaust hood. Cover the
fixation tank. Change the solution after each 24 hour
15 interval.
Measure 7.68 liters of demineralized water and
place in a clean carboy. Weigh out 112.0 g of sodium
citrate and add to the carboy. Weigh out 4.0 g of citric
acid and add to the carboy. Place the carboy on a stir
ZO plate and mix until all solids have been dissolved.
Citrate concentration should be 50 mM, consisting of
95.2% sodium citrate and 4.8o citric acid.
.: Measure 320 ml of 50% glutaraldehyde and add to
the citrate buffer while it is still on the stir plate.
25 Allow the solution to equilibrate for 10-15 minutes.
Test pH and adjust as required to 6.40 +/- 0.05 using 6M
NaOH or 6M_ HC1. Transfer glutaraldehyde solution to a
clean fixation tank prior to the sterilization process.
After approximately 120 hours of fixation,
.. 30 transfer the grafts to a 2o glutaraldehyde solution.
Place fixation tank under the exhaust hood. Cover the
fixation tank. Allow grafts to remain in the 2%
glutaraldehyde for 4-5 hours.
Example 10.
Biological tissues can be produced as follows:
2169381
31
1. individual tissue valves are exposed to 0.01%,
0.02%, 0.03%, 0.04x, 0.05%, 0.06%, 0.07%, 0.08%, or 0.09%
glutaraldehyde, buffered in either citrate (at pFi 6.4) or
FiEPES (at pIi 7.4) , at 20°C for 2, 4, 5, 7, 9; or 10 days.
The crosslinked biological tissue can then be
initially sterilized in a mufti-component sterilant of 2%
glutaraldehyde, 3% formaldehyde, and 20% ethanol in an
aqueous buffer. The initially sterilized biological
tissue may then be subjected to ethanol extraction (60%
l0 ethanol), optionally including O.1M A1C13, and a 24 hour
wash in saline.
The biological tissue is then packaged in a saline
solution and subjected to terminal sterilization by
exposing the tissue to e-beam radiation.
While the invention has bean described in some
detail by way of illustration and example, it should be
understood that the invention is susceptible to various
modifications and alternative forms, and is not
restricted to the specific embodiments set forth. It
should be understood that these specific embodi:~ents are
not intended to limit the invention but; on tze contrary;
the intention is to cover all modifications, equivalents,
. and alternatives falling within the spirit and scope of
the invention.
