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Description  |
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The present invention relates to a process for fixing biological tissue
prior to implantation into a mammal, and more particularly to a fixation
process which creates amide linkages between and within the molecules of
the biological tissue thereby producing tissues resistant to
calcification. In addition, the resulting tissue is not toxic and does not
elicit inflammatory responses after implantation.
BACKGROUND OF THE INVENTION
The surgical implantation of prosthetic devices (prostheses) into humans
and other mammals has been carried out in recent years with increasing
frequency. Such prostheses include, by way of illustration only, heart
valves, vascular grafts, urinary bladders, left ventricular-assist
devices, hip prostheses, silastic breast implants, tendon prostheses, and
the like. They may be constructed from natural tissues, inorganic
materials, synthetic polymers, or combinations thereof. By way of
illustration, mechanical heart valve prostheses typically are composed of
rigid materials, such as polymers, carbons and metals and employ one or
more occluders which respond passively with changes in intracardiac
pressure or flow. Natural tissue heart valve prostheses, on the other
hand, typically are fabricated from either porcine aortic valves or bovine
pericardium; in either case, the tissue is normally pretreated with
glutaraldehyde and then sewn onto a flexible metallic alloy or polymeric
stent which subsequently is covered with a poly(ethylene terephthalate)
cloth sewing ring covering. Typically, the assembled prostheses (known to
those of skill in the art as bioprostheses) are then stored in 0.2 percent
glutaraldehyde.
Bioprostheses, that is prostheses derived from natural tissues, may be
preferred over mechanical devices because of certain significant clinical
advantages; for example, bioprostheses generally do not require routine
anticoagulation. Moreover, when they fail, they usually exhibit a gradual
deterioration which can extend over a period of months, or even years.
Mechanical devices, on the other hand, generally require regular
anticoagulation therapy and very occasionally undergo catastrophic
failure; however, they do have other advantageous features.
Bioprostheses must be treated, prior to implantation into an animal
different from the donor animal, in order to stabilize the tissue. This
process of stabilization is known in the art as fixation. In 1968, Nimni
et al. demonstrated that collagenous materials, the major component of
bioprostheses, can be fixed by treating them with aldehydes. (Nimni et
al., J. Biol. Chem. 243:1457-1466 (1968).) Later, it was discovered that,
of various aldehydes tested, glutaraldehyde best retards degeneration of
collagenous tissue. (Nimni et al., J. Biomed. Mater. Res. 21:741-771
(1987); Woodroof, E. A., J. Bioeng. 2:1 (1978).) Generally, the fixation
process operates by blocking reactive molecules on the surface of and
within the donor tissue, thereby rendering it substantially non-antigenic
and suitable for implantation. Thus, the process of
glutaraldehyde-fixation has been and continues to be applied to most all
varieties of experimental and clinical bioprostheses.
Early experimental and clinical studies of glutaraldehyde-preserved
bioprostheses were of bioprosthetic heart valves. The data compiled from
these early studies demonstrated the excellent biomechanical properties,
high resistance to enzymatic degradation, excellent hemodynamic properties
and minimal thrombogenicity of the glutaraldehyde-preserved heart valve.
However, follow-up clinical studies questioned the long-term durability of
glutaraldehyde-fixed valves due to a variety of problems such as cuspal
infection, low-grade immune reactions, severe calcification, stenosis and
biodegradation. Further, it was discovered that the
glutaraldehyde-fixation process induces toxic reactions due to the slow
release of glutaraldehyde from the implanted tissue. These toxic reactions
may be partially responsible for the immune reactions and the lack of
endothelial cell coverage also found in these implants.
Calcification, which causes prosthesis degeneration, is an especially
significant disadvantage to the use of tissue-derived prostheses. Indeed,
cuspal calcification accounts for over 60 percent of the failures of
cardiac bioprosthetic valve implants, such failures being substantially
more frequent in children than in adults. Despite the clinical importance
of the problem, the pathogenesis of calcification is incompletely
understood. It seems that calcification is related to the extent of
glutaraldehyde-induced cross-links and results from intrinsic and
extrinsic mineralization in and on the surface of the bioprosthesis.
(Schoen, F., J. Card. Surg. 2(1):65 (1987).) Further there is evidence of
a specific calcium-binding amino acid, laid down after implantation of
glutaraldehyde-preserved porcine bioprostheses, which has been postulated
to play a role in calcification.(U.S. Pat. No. 4,770,665)
Efforts at retarding the calcification of bioprosthetic tissue have been
numerous in recent years. The techniques resulting from these efforts may
be broadly divided into two categories; those involving the pre- or
post-treatment of glutaraldehyde-fixed tissue with one or more compounds
that inhibit calcification (or modify the fixed tissue to be less prone to
calcification) and those involving the fixation of the tissue with
compounds other than glutaraldehyde, thereby reducing calcification.
The former category of techniques includes, but is not limited to,
treatment with such compounds as:
a) detergent or surfactant, after glutaraldehyde fixation;
b) diphosphonates, covalently bound to the glutaraldehyde-fixed tissue or
administered via injection to the recipient of the bioprosthesis or
site-specifically delivered via an osmotic pump or controlled-release
matrix;
c) amino-substituted aliphatic carboxylic acid, covalently bound after
glutaraldehyde-fixation;
d) sulfated polysaccharides, especially chondroitin sulfate, after
glutaraldehyde fixation and preferably followed by treatment with other
matrix-stabilizing materials;
e) ferric or stannic salts, either before or after glutaraldehyde fixation;
f) polymers, especially elastomeric polymers, incorporated into the
glutaraldehyde-fixed tissue; or
g) water-soluble solutions of a phosphate ester or a quaternary ammonium
salt or a sulfated higher aliphatic alcohol, after
glutaraldehyde-fixation.
The latter category of techniques for reducing the calcification of
bioprosthetic tissue, i.e., techniques involving the fixation of the
tissue with compounds other than glutaraldehyde, includes but is not
limited to, the following:
a) treatment by soaking the bioprosthetic tissue in an aqueous solution of
high osmolality containing a photo-oxidative catalyst and then exposing
said tissue to light, thereby fixing the tissue via photo-oxidization; and
b) fixation via treatment with a polyepoxy compound, such as polyglycidyl
ether (polyepoxy) compound.
In most cases, investigations related to glutaraldehyde-associated
symptomatology have been limited to specific problems such as
calcification and have not addressed the entire spectrum of symptoms.
Thus, while the problem of calcification of glutaraldehyde-fixed
bioprostheses has received a great deal of attention, the proposed
solutions have generally failed to address any other complications
presented by the presence of glutaraldehyde, such as toxicity, immune
reactions and degeneration. Glutaraldehyde released from the tissue is
cytotoxic and prevents the formation of endothelial cell growth on the
bioprosthesis necessary for long-term durability. This persistent damage
to the implant and surrounding tissue due to the long-term slow release of
glutaraldehyde may be fully eradicated only by using a fixation process
that does not include glutaraldehyde. The complexity and gravity of the
clinical problems resulting from glutaraldehyde-preserved bioprostheses
warrant the search for an alternative fixation method.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method for the
non-glutaraldehyde fixation of a prosthesis to be implanted in a mammal
whereby said prosthesis is fixed by forming amide linkages between and
within the molecules of the prosthetic tissue. Also, the invention
provides prostheses, suitable for implantation in a mammal, made by the
aforesaid method.
In one preferred embodiment, di- or tri- carboxylic acids and di- or
tri-amines of about six to about eight carbon atoms in length, are used,
in a sequential manner, to form amide cross-links. In a particularly
preferred embodiment, suberic acid, a di-carboxylic acid, and 1,6-hexane
diamine are used to form the amide linkages, resulting in cross-linking
chains of about six to about twenty-four carbon atoms in length between
and within the molecules of the tissue of the prosthesis.
The method of the present invention is useful for preventing or retarding
the calcification of a prosthesis implanted in a mammal, such as a human,
and it provides a product that does not cause an inflammatory response in
the body and is nontoxic. It has particular application with respect to
those prostheses which are normally fixed using glutaraldehyde and more
particularly, to those prostheses which are especially susceptible to
degeneration as a result of calcification.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "prosthesis" is meant to include any prosthesis
which is derived in whole or part from animal or other organic tissue and
which is to be implanted in a mammal. Thus, the term generally includes
bioprostheses, such as heart valves and other heart components, vascular
replacements or grafts, heart replacements, urinary tract and bladder
replacements, bowel and tissue resections in general, and the like.
However, it will be recognized by those having ordinary skill in this art
that the present invention may be of most importance in relation to
prostheses for which glutaraldehyde release and resultant toxicity,
degeneration and/or calcification after implantation has been a clinical
problem. Thus, while the present invention can be used with essentially
any bioprosthesis, it may not be as beneficial for a prosthesis which is
not normally fixed using glutaraldehyde or is not otherwise likely to
suffer degeneration or malfunction as a result of mineralization.
The fixation process described herein relies on the availability of free
active moieties on and within the prosthetic tissue that are capable of
forming amide bonds with the cross-linking agent(s); for example, carboxyl
and amine moieties. Thus, the prosthesis will be one which is made from
natural tissues, including but not limited to bovine, ovine, porcine and
possibly even human tissue. Other natural materials, well known to those
having ordinary skill in the art, also can be used.
The term "cross-link" is defined as understood by those of skill in the
art. Generally, where biological tissue is concerned, cross-linking refers
to the process of forming covalent bonds (or cross-links) either directly
between free active moieties on or within the tissue or between the free
active moieties of the tissue and one or more compounds (or cross-linking
agents), in such a manner as to leave few or no active moieties on or
within the tissue. This cross-linking process "fixes" or stabilizes the
tissue by making the tissue less antigenic and thus less susceptible to
degradation than before the process.
Thus the term "fixation" as used herein, and as generally understood by
those of skill in the art, refers to the process of treating biological
tissue in order to stabilize it for implantation in a host animal
different from the donor individual. Currently, most bioprosthetic tissue
is fixed via treatment with glutaraldehyde.
In general, a "cross-linking agent or reagent", as used herein, is a
compound capable of covalently binding to the free active moieties of
prosthetic tissue and/or to other cross-linking agents in such a manner as
to result in cross-links between and within the molecules of the
prosthetic tissue and between the molecules of the prosthetic tissue and
the agent, thereby fixing said tissue. The cross-linking agent(s) is
selected in such a way as to maximize fixation of the tissue being treated
while minimizing the risk of damage to the prosthesis during treatment and
minimizing the risks, such as of toxicity, inflammation, calcification,
etc., to the host animal in whom the treated prosthesis is to be
implanted. The cross-linking agents are preferably water-soluble so that
aqueous buffers may be utilized thereby minimizing the risk of damage to
the prosthesis during the fixation process.
Further, the cross-linking agents or reagents have at least two reactive
moieties sufficiently distant from each other to permit covalent binding,
at at least two locations, between itself and the prosthetic tissue and/or
another cross-linking agent. Preferably, the reactive moieties on any one
cross-linking agent are the same and bind via formation of amide bonds.
Also preferably, the cross-linking agents are chains from about 4 to about
24 carbon atoms in length and most preferably from about 6 to 8 carbon
atoms in length. The reactive moieties are preferably separated by a chain
of at least 4 carbon atoms (more preferably at least 6) and are preferably
located at the respective ends of the longest carbon chain. One or more
cross-linking agents may be used, and preferably, at least two different
agents will be used. In one preferred embodiment, either a di- or
tri-amine and either a di- or tri-carboxylic acid are used as the two
cross-linking agents.
The cross-linking agents may be straight-chained or branched-chained,
appropriately substituted compounds. Preferably they are straight chains
having the reactive moieties or groups at each terminus. Except for the
substituent through which covalent linking to the prosthesis or another
cross-linking agent is achieved, the nature and number of the substituents
are not critical, provided that they do not induce calcification or other
adverse physiological effects upon implantation; do not create
non-water-soluble or toxic by-products; and do not adversely effect the
compound's water solubility. Preferably, any such substituents should
assist to stabilize the tissue.
The concentration of each cross-linking agent can vary and will depend on
the nature of the cross-linking agent, for example, the efficiency with
which it binds to the prosthetic tissue and/or another cross-linking
agent. In certain preferred embodiments, concentrations ranging from about
5 mM (millimolar) to about 20 mM are used; however, one skilled in the art
can readily determine appropriate concentrations for each cross-linking
agent.
The terms "coupling agent" and "coupling enhancer", as used herein, refer
to reagents that respectively, initiate and enhance the cross-linking
reaction between cross-linking agents and/or between prosthetic tissue and
a cross-linking agent. Those of skill in the art will be familiar with
which such reagents are most effective with which cross-linking agents. In
a preferred embodiment, 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide
hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) are the
coupling agent and coupling enhancer, respectively. The concentration of
the coupling agent and coupling enhancer can vary and will depend on such
things as the cross-linking agent employed. Appropriate concentrations are
readily determinable by those of skill in the art.
Reaction conditions for the cross-linking of the prosthesis tissue may
vary, depending on the cross-linking agents and cross-linking chemistry
employed. In general, the cross-linking process is carried out in an
aqueous solution buffered at a suitable pH. A suitable buffer is used, and
buffers from among those well known to those of ordinary skill in this art
are chosen so as to best suit the cross-linking agents being employed.
Examples of suitable buffers include, but are not limited to,
N-2-Hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES) and
3-(N-morpholino)propanesulfonic acid (MOPS), and the like. For additional
details on buffers, see, e.g., Gerald D. Fasman, Editor, "CRC Handbook of
Biochemistry and Molecular Biology," 3rd Edition, Physical and Chemical
Data Volume I, CRC Press, Inc., Boca Raton, Fla., 1976, pp 354-377. The
buffer concentration is not critical and can vary fairly widely.
The pH of the buffer also can vary, again depending upon the cross-linking
agents to be employed. In preferred embodiments, the buffer concentration
and pH are chosen to be the least harmful to the prosthetic tissue and
cross-linking agents and the most effective for the cross-linking
reaction; for example, a preferred embodiment employs a HEPES buffer at a
pH from about 6.5 to about 7.4. All solutions are filtered before use, for
example, through 0.45 .mu.m Acrodisc.TM. filters.
The prosthetic tissue to be fixed by the cross-linking method herein
described is preferably freshly obtained from the donor animal and
immediately processed. For example, the tissue is rinsed or washed in an
appropriate aqueous solution to remove red blood cells and other
contaminating debris. In preferred embodiments, ice cold 0.85% saline
solution is used. Those of skill in the art will readily recognize
alternative, equivalent solutions for this cleaning. If the tissue is
stored, for example overnight, it is stored in an appropriate buffer as
described further below, at an appropriate, lower temperature such as
about 4.degree. C.
After processing as described above, the prosthetic tissue is incubated
with a cross-linking agent, a coupling agent and a coupling enhancer.
Incubation time can vary and will depend upon such things as the
particular cross-linking agents, coupling agent and coupling enhancer
employed, as well as the conditions of cross-linking such as
concentrations of agents, pH and temperature. In preferred embodiments,
the incubation time is about 48 hours. The cross-linking agent is chosen
as previously described, preferably, such that the active moieties on the
agent readily covalently bind, via amide bonds, to the prosthetic tissue.
For example a cross-linking agent with either multiple active carboxyl or
multiple amine moieties is particularly preferred. Because the reactions
described herein are for the cross-linking of bioprosthetic tissue, the
temperature of reaction typically will not exceed 40.degree. C. nor fall
below 0.degree. C., and preferably, reaction is carried out at room
temperature.
This first coupling reaction results in a cross-linking agent being
covalently bound to the prosthetic tissue by one or more of its reactive
moieties. Most molecules of cross-linking agent will be bound (or
anchored) to the tissue via one reactive moiety with the other moiety(s)
being either bound to the tissue or unbound and thus free for further
reaction. After a sufficient incubation time as described above, the
tissue is rinsed or washed in aqueous buffer to remove non-reacted and
water-soluble by-products. Appropriate buffers are used as previously
described and as understood by those of skill in the art. The
bioprosthetic tissue is not permitted to dry, rather it is maintained in
the buffer solution.
Further cross-linking is accomplished in the same manner as just described
by using a second cross-linking agent that is capable of covalently
binding, preferably via amide bond formation, to the first cross-linking
agent and optionally to the prosthetic tissue. This results in the second
cross-linking agent being bound, via one or more of its reactive moieties,
to an unbound moiety of the first cross-linking agent or to the prosthetic
tissue or to both. Thus, carbon atom chains of various lengths are formed
between and within the prosthetic tissue. Additional coupling reactions
are performed as necessary to effectively fix the prosthetic tissue. In
preferred embodiments, the fixation process is considered to be complete
after three such coupling reactions. In a particularly preferred
embodiment, the third cross-linking agent which is used is of the same
composition as the first cross-linking agent.
The method of fixation described herein, whereby water-soluble
cross-linking agents of between about 4 and about 24 carbon atoms in
length are coupled to bioprosthetic tissue, results in a myriad of links
(or bridges) of cross-linking agents between and within the molecules of
the prosthetic tissue having reactive moieties. Fixation is complete when
few if any reactive moieties remain unreacted on the tissue's surface or
just within the tissue. Some short links will be comprised of a single
molecule of a cross-linking agent bridging two reactive moieties located
on or within the prosthetic tissue; however, it is believed that a major
portion of the links in the prosthetic tissue will be comprised of two or
more cross-linking agents connected to each other.
The preferred embodiments of cross-linking agents have carbon chains which
contain multiple carboxylic acid or amine reactive moieties which form
amide bonds with one another. The prosthetic tissue is also capable of
forming amide bonds with both of these preferred cross-linking moieties
because of the presence of reactive carboxyl and amine groups on and
within the tissue.
The present invention is further described by the examples which follow.
Such examples, however, are not to be construed as limiting in any way
either the spirit or the scope of the present invention. In the examples,
percentages are percents by volume or of weight per unit volume, e.g.,
grams per liter, and all temperatures are in degrees Celsius, unless
otherwise specified.
EXAMPLE 1
A process embodying feature of the present invention is illustrated by the
formation of amide linkages between and within porcine aortic valve tissue
by the sequential coupling of a di-carboxylic acid and a diamine to
molecules forming this tissue.
I. Preparation of Porcine Tissue
Fresh porcine hearts were obtained from a slaughterhouse, on ice, 24 hours
after slaughter and immediately processed. The aortic valve leaflets and
wall specimens (1.times.3.5 cm coupons) were dissected and rinsed 6 times
in ice-cold 0.85% saline to remove red blood cells and other debris. They
were then stored overnight at 4.degree. C. in 10 mM HEPES, 0.85% NaCl, pH
6.5 (HEPES buffer).
II. Cross-linking of Porcine Aortic Valve Tissue
A. Addition of First Cross-linking Agent
Twenty-five leaflets and three wall coupons were transferred from the HEPES
buffer to 75 ml of the first cross-linking agent, 10 mM suberic acid (98%,
purchased from Aldrich), prepared in HEPES buffer. A twenty-five ml
solution of a coupling agent, 200 mM 1-ethyl-3(3-dimethyl aminopropyl)
carbodiimide hydrochloride (EDC), together with a coupling enhancer, 10 mM
N-hydroxysulfosuccinimide (sulfo-NHS) (obtained from Sigma and Pierce,
respectively) in HEPES buffer, prepared immediately prior to use, was then
added to the solution to initiate the cross-linking reaction. The jar
containing the reagents, leaflets and wall coupons was incubated for 48
hours at room temperature. The samples were then rinsed once with 50 ml of
HEPES buffer to eliminate non-reacted and water soluble by-products of the
reaction. At this point, the suberic acid is covalently bound to free
amine groups of the collagenous tissue. Some of the acid molecules may
bridge two tissue amine groups, but most of them will be anchored to one
amine moiety, leaving one carboxyl group free and available for further
reaction.
B. Addition of Second Cross-Linking Agent
Next, the valve leaflets and wall coupons were transferred to a 75 ml
solution of the second cross-linking agent, 15 mM 1,6-hexane diamine (98%,
purchased from Aldrich), prepared in HEPES buffer. The cross-linking
reaction was carried out as described above; that is, addition of
EDC/sulfo-NHS, incubation for 48 hours at room temperature, and then a
single HEPES buffer rinse. This second coupling reaction results in chains
of six, eight, fourteen or even twenty-two carbon atoms in length between
and within the collagenous molecules of the tissue samples, with a large
portion of the second cross-linking agent having one amine group free for
further reaction.
C. Addition of Third Cross-Linking Agent
As the third and final cross-linking agent, 10 mM suberic acid was again
applied to the leaflets and wall coupons, using the same conditions as
described for the first coupling reaction. Once this final coupling
reaction was complete, the tissue samples were rinsed in HEPES buffer and
stored until use at room temperature in HEPES buffer pH 7.4 containing 20%
isopropanol.
EXAMPLE 2
An alternative fixation process is carried out by the formation of amide
linkages between and within porcine aortic heart valve tissue by the
initial coupling of a diamine, followed by the coupling of a di-carboxylic
acid to this tissue. Twenty-five leaflets and three wall coupons were
obtained from fresh porcine hearts, and treated as described in Example 1,
save that the cross-linking agent employed, in steps IIA. and C. was
1,6-hexane diamine, and the cross-linking agent employed in step IIB. was
suberic acid.
EXAMPLE 3
A further alternative fixation process is carried out by the formation of
amide linkages between and within porcine aortic heart valve tissue by the
initial coupling of a diamine followed by coupling of a tri-carboxylic
acid to this tissue. Twenty-five leaflets and three wall coupons were
obtained from fresh porcine hearts and treated as described in Example 2,
save that, instead of suberic acid, 7 mM 1,3,5-benzenetricarboxylic acid
(98%, purchased from Aldrich), prepared in HEPES buffer was used as the
second cross-linking agent.
EXAMPLE 4
A still further fixation process is carried out by the formation of amide
linkages between and within porcine aortic heart valve tissue by the
initial coupling of a tri-carboxylic acid followed by coupling of a
diamine to this tissue. Twenty-five leaflets and three wall coupons were
obtained from fresh porcine hearts and treated as described in Example 1,
save that as the first and third cross-linking agents, in steps IIA. and
C., 7 mM 1,3,5-benzenetricarboxylic acid (98%, purchased from Aldrich),
prepared in HEPES buffer, was employed.
Characterization and Comparison of Treated Tissue and Various Control
Tissues
The porcine aortic valve tissue treated as described in Examples 1 to 4,
inclusive, together with two controls and a comparison group, were
characterized and compared. The control groups included an "EDC control"
of porcine aortic valve tissue treated as described in Example 1, save
that HEPES buffer was used in place of the cross-linking agents, and a
"fresh tissue control" of porcine aortic valve tissue stored in HEPES
buffer containing 20% isopropanol. Also, a "glutaraldehyde comparison" of
tissue dissected from glutaraldehyde-fixed porcine aortic valves was used
(provided by Medtronic, Heart Valves Division, Calif.).
I. Thermal Denaturation
A. Method
Three leaflets from each of Examples 1-4, the glutaraldehyde comparison and
the above-described two controls were secured between one fixed and one
rotating alligator clip attached to a reading needle providing a 5-fold
length amplification. They were then immersed in distilled water at
45.degree. C. The water temperature was then increased at a rate of
1.5.degree. C. per minute. The temperature at which the tissue started to
contract was recorded as the shrinkage temperature and expressed as mean
.+-.SEM.degree. C. shrinkage temperature.
B. Results
The shrinkage temperatures of leaflets fixed using processes set forth in
Examples 1 through 4 as well as the shrinkage temperature of the EDC
control were all higher than the shrinkage temperature of the
glutaraldehyde comparison, which was higher than the shrinkage temperature
of the fresh tissue control. Shrinkage temperature (or point of thermal
denaturation) is directly related to the density of cross-linking on and
within the molecules of the tissue. Thus, fixation of the tissue by the
process of Examples 1-4 confers at least as great if not greater
cross-linking density to the tissue than does fixation by the
glutaraldehyde method. The shrinkage temperature of the EDC control is as
high as that of glutaraldehyde and suggests that EDC alone induces
cross-linking between close residual carboxyl and amine moieties located
within the tissue itself.
II. Residual Amine Test
A. Methods
Three half leaflets and 3 wall coupons from each of Examples 1-4, the
glutaraldehyde comparison and the controls were incubated at 95.degree. C.
for 20 minutes in 1 ml of ninhydrin in citrate buffer, pH 5.0. They were
cooled to room temperature, then removed from their incubation solutions,
dried and weighed. The incubation solutions were diluted with 1 ml of 50%
(v/v) isopropanol in distilled water. The optical absorption was
determined at 570 nm using a Beckman DB-G spectrophotometer. A standard
curve was established using L-norleucine at concentrations ranging from 0
to 100 nM/ml. The results are expressed as nanomoles of 1-norleucine amine
equivalent per mg of dry tissue and are compared.
B. Results
The relative differences between conditions were similar for the cusps and
the walls. The level of residual amines was significantly lower for all
fixed tissue (Examples 1-4, glutaraldehyde comparison and EDC control)
than for the fresh tissue. The levels were not significantly different
between the glutaraldehyde comparison and the samples from Examples 1 and
4. A higher level of residual amines was observed in the samples from
Examples 2 and 3 as compared to the glutaraldehyde comparison. This was
probably due to the residual amines resulting from the diamine treatment
of the third coupling reaction. The low level of residual amines observed
in the EDC control, as compared with the fresh tissue control, was a
reflection of the fact that treatment of the tissue with EDC in the
absence of cross-linking agents results in the modification of
approximately 63% of the amines and thus allows partial cross-linking of
the tissue, as was also reflected in the shrinkage temperature study,
above. Thus, as a measurement of the degree of fixation of the tissue, the
residual amine test suggests that the fixation processes in Examples 1-4
are as efficient as the glutaraldehyde method.
III. Collagenase Digestion
A. Methods
A collagenase solution was prepared by dissolving 5 mg collagenase, 180 mg
CaCl.sub.2.2H.sub.2 O and 65 mg sodium azide in 130 ml of HEPES buffer, pH
7.4. Three half-leaflets and three wall coupons from each of Examples 1-4,
the glutaraldehyde comparison and the controls were dried for 1 hour at
room temperature, individually minced, weighed and incubated in 3 ml of
the collagenase solution at 37.degree. C. for 72 hours in an orbital
shaker set at 150 rpm. The levels of amines released from the samples were
determined by the ninhydrin test described in the residual amine test
above, using 0.1 ml of collagenase solution free of particulate material
in 1 ml of ninhydrin in citrate buffer solution pH 5.0. The results are
expressed as nanomoles of 1-norleucine amine equivalent per mg of dry
tissue and compared.
B. Results
The fresh tissue control sample showed in excess of 350 nanomoles amine
equivalent per mg dry tissue released and had no resistance to collagenase
whereas the samples from each of Examples 1-4, as well as the EDC control
and glutaraldehyde comparison, were fully resistant to digestion. Thus,
the fixation processes of Examples 1-4 result in tissue that is as
resistant to collagenase digestion as the glutaraldehyde-fixed tissue.
IV. Protease Digestion
A. Methods
A protease solution was prepared by dissolving 75 mg of protease and 75 mg
CaCl.sub.2.2H.sub.2 O in 150 ml of HEPES buffer pH 7.4, containing 50 mM
glycine. Three half-leaflets and three wall coupons from each of the four
examples and the controls were blotted and weighed. In order to obtain the
dry weight of the samples prior to protease digestion, a piece (25%) of
each blotted sample was cut, dried and weighed. The ratio of blotted
weight/dry weight was then used to calculate the dry weight of the
specimens prior to protease digestion. The remaining 75% of the samples
were incubated in 3 ml of protease solution at 50.degree. C. for 22 hours.
They were then removed from the protease solution, dried and weighed. The
weight loss corresponds to proteolytic degradation, and the results are
expressed as % weight decrease after 22 hours of incubation.
B. Results
The fresh tissue control leaflets and walls were completely digested by the
nonspecific protease during 22 hours of incubation. The leaflets from
Examples 1-4 inclusive and the EDC controls resisted digestion as well as
the glutaraldehyde-fixed leaflets. The walls from Examples 1-4 and the EDC
controls showed greater resistance to protease digestion than did the
glutaraldehyde comparison walls. Therefore, based on this protease
digestion test, tissues fixed according to Examples 1-4 are at least as
well, if not better, cross-linked than glutaraldehyde-fixed tissues.
V. Calcification
An important advantage offered by the present invention is illustrated by a
comparison of the calcium levels of the treated and control tissues after
implantation subdermally in rats.
A. Method
Twelve half-leaflets and three wall coupons from each of Examples 1-4 and
from the glutaraldehyde-fixed and EDC control groups were washed 3 times
in saline and implanted subdermally in the abdominal area (4 implants per
animal) of 3 week-old male Sprague Dawley rats. Six half-leaflets and the
3 wall coupons per test group were retrieved after 4 weeks, and the
remaining 6 half-leaflets per group were retrieved after 8 weeks. The
surrounding tissue capsule was removed. The samples were washed 5 times in
distilled water, lyophilized, weighed and hydrolyzed in 1 ml of ultrapure
6N HCl at 85.degree. C. for 24 hours. The hydrolysates were dried under
vacuum and the residues resuspended in 0.3N HCl. Calcium levels were
determined by the Inductively Coupled Plasma analysis method using a
Perkin Elner 8000 ICP spectrophotometer at Georgia Tech Research
Institute. The results are expressed as milligrams of calcium per gram of
dry tissue.
B. Results
The following Table 1 illustrates the significant difference between the
minimal calcification of the tissues fixed using the processes of Examples
1-4 and the high degree of calcification observed in the tissues fixed
using the glutaraldehyde method, with the approximate average values for
each being expressed.
TABLE 1
______________________________________
Mean .+-. SEM
(miligrams calcium/gram dry tissue)
Leaflets Leaflets Walls
Method of implanted implanted implanted
Fixation 4 weeks 8 weeks 4 weeks
______________________________________
Glutaraldehyde
204 .+-. 10
230 .+-. 34 130 .+-. 8
Example 1 9.5 .+-. 4.8
31 .+-. 21 35 .+-. 4
Example 2 8.1 .+-. 5 19 .+-. 18 26 .+-. 14
Example 3 4.2 .+-. 2,2
36 .+-. 13 33 .+-. 5
Example 4 16 .+-. 6 6 .+-. 4 22 .+-. 8
EDC control
25 .+-. 15 21 .+-. 19 40 .+-. 8
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