Note: Descriptions are shown in the official language in which they were submitted.
COATING FOR BIOPROSTHETIC DEVICE
AND
METHOD OF MAKIN~ SAME
BACKGROUND OF THE INVENTION
For many years, a variety of animal tissues,
as well as some synthetic polymers~ have been used
to make prosthetic devices for surgical implantation
into human beings and other animals. HoweverJ because
these devices are different on a molecular level from
the host organism s own tissue, they usually elicit
a wide variety of reactions in the host. The response
is manifested by a low-grade, rapid deterioration of
~he transplant, which in turnt mandates additional
surgery.
To improve the longevity of transplanted
devices, a number of remedies have been proposed.
In the processing of natural tissues~ a common stabil-
zation technique involves treatment with tanning
agents~ such as formaldehyde. Glutaraldehyde, a well
known cross-linking agent, has also been used with
success in this regard. In fact, a number of studies
have shown that heart valves treated with
glutaraldehyde can remain functional in situ for many
years. ~owever, recent research has indicated that
such glutaraldehyde preserved implantations can still
elicit signiicant host reactions, including
calcification, fibrin deposition and an anaphylactic
response r ( For example, see Slanczka, D.J. and
Bajpai, P.K., "Immunogenicity of Glutaraldehyde-
treated Porcine Heart Valves", IRCS Medical Science:
Bio-Technology; Cardiovascular System; Immunology
and Allergy; Pathology; Surgery and Transplantation;
' .:,
~r~,
:~2~
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6, 421 (1978).)
It has also been theorized that natural
prosthetics may be biodegradable~ and thus labile even
after short placement periods. In vitro enzyme
degradation of the tissues prior to implantation
has been utilized to minimize this obstacle, but
this degradation is not totally effective in
mitigating the antigenic response; and moreover~ the
tissue can lose significant portions of its inherent
structural framework, which can cause further
mechanical weakening of the entire device.
Although considerable success has been
achieved by implanting synthetic devices instead of
natural devices, at present, they also present
significant difficulties. There is a substantial
biological failure rate among these devices due to
incompatibility with biological tissues. After
removal of the implant, fibrin layering, aneurysm
formation, lipid deposition and many clinical mal-
func~ions have been noted.
A further problem, common to many of thesynthetic and natural prosthetics alike, is minimal
flexibility~ Glutaraldehyde-treated natural devices
are often cross-linked to such a degree that much of
their natural flexibility is lost, and after prolonged
periods of implantation, brittleness often becomes
even more pronounced. Similarly, synthetic devices
generally become increasingly hardened after prolonged
implantation.
Therefore, there is a recognized need for
an improved treatment of prosthetic devices prior to
implantation, which will render these devices more
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durable, yet minimize negative host responses. The
present invention fulfills this need.
SUMMARY OF THE INVENTION
In accordance with one aspect of the
invention, a coating for heart valves and other pro-
sthetic devices is provided that has greatly improvedbiophysical stability after the device is implanted
in a host organism. Through the formation of a three-
dimensional cross-linked matrix primarily composed of
a calcification inhibitor covalently bound to
accessible regions of the device, a substantially
non-antigenic bioprosthesis with minimum calcification
sites may be produced.
Suitable calcification inhibitors include
natural protein polysaccharides, such as chondroitin
sulfates and hyaluronate. Generally, sulfated
polysaccharides are preferred, but diphosphonates,
phosphoproteins, dyes, such as alzarin red S and
methylene blue, and other polyanions may be used.
The incorporation of other agents into the
matrix can further enhance long term survival of the
implanted device. Specifically, bridging agents, such
as diamines, that create additional cross-linking
sites and additional covalent binding sites for
attaching other specified materials, such as
antithrombogens, may be bound to the matrix~ Also,
the presence of materials that fill the interstitial
gaps in the matrix can provide greater stability by
limiting nucleation and the growth of hydroxyapatite
crystals.
~za~s~
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Another aspect of the invention is a process
for treating bioprosthetics to provide a coating, such
as described above, for improved stability after
implantation. The method, which can utilize the
compounds described above, comprises the steps of:
harvesting tissue from an organism; intitiating a
number of covalent cross-links~ preferably with
glutaraldehyde, in the protein structure of the
tissue sufficient to protect the tissue from initial
losses in structural integrity; soaking the tissue
in calcification inhibitor; covalently binding the
calcification inhibitor to the tissue, preferably with
a carbodiimide; and sterilizing. Additional steps
may include the covalent binding of bridging agents,
such as diamines, antithrombogenic agents and gap
filling materials to the tissue. The treatment is
particularly useful for rendering animal connective
tissues, such as mammalian heart valves and blood
vessels, substantially water insoloble and less likely
to initiate calcification than natural tissue or
tanned tissue.
DETAILED DESCRIPTION OF THE INVENTION
.. .. . .. _ .
AND
PREFERRED EMBODIMENTS
Exemplary starting materials useful in
practicing the invention include- animal tissues
of diverse origin, e.g. heart valves, blood vessels,
peracardia, dura mater, ligaments, tendons, and other
collagen-rich tissues, as well as reconstituted or
native collagen fibers and other materials with
accessible cross-linking sites. Assuming tissues are
used, they are first cleaned from adherent fat or
loose connective tissue as soon as possible after
harvesting. Immediately thereafter, they are placed
in a balanced electrolyte solution that is calciurn-
free and buffered at a neutral ph with a phosphate
buffer. This solution, kept cool (4-8 degrees Centi-
grade), contains a calcium chelator, such as EDTA-Na
at about 0.05 molar concentration, to sequester calci-
um present in the tissue.
The following steps are then utilized to
adequately cross-link and modify the tissue in this
exemplary process:
1) Immediately after harvesting and
cleaning, the tissue is placed in a
solution containing 0.05 wt. % glutaral-
dehyde buffered with phosphate at pH
7.0, and made isotonic with a calcium
free, balanced electrolyte solution.
This causes partial cro 5 S- linking of
the collagen and the protein-like
compounds naturally associated with
it (called protein-polysacchrides) and
is performed to prevent swelling and
distortion of the ultrastructure of the
connective tissue.
2) The tissue is then placed in a solution
containing a calcification lnhibitor,
preferably chondroitin sulfate at a
concentration of about 0.5 to about 5
wt. ~, preferably about 1.0 wt. ~.
Chondroitin sulfate is available com-
mercially or may be prepared from a
variety of cartilagenous sources. In
some instances~ it may be desirable to
use the protein-polysaccharides associ-
ated with collagen in natural tissues.
}5~
These include chondroitin-6-sulfate,
chondroitin-4-sulfate and hyaluronate.
Generally~ polysaccharides of the
chondroitin sulfate variety that are
rich in weak negative charges tcarboxyl
groups) and in strong negative charges
~sulfate groups)~ such as sulfated
polysaccharides, are preferred. Other
substances that are known inhibitors of
calcification include diphosphonates,
which are characterized by the presence
of a P-C-P or a P-N-P bond. It is
theorized that P-C-P and P-N-P bonds are
not "bio-degradable" and are t therefore,
very stable in tissues. A typical
diphosphonate is 3-amino-1-hydroxy-
propane 1, l-diphosphonic acid. Other
diphosphonates with active amino or
carboxyl groups can easily be attached
by covalent bonds and act as inhibitors
of calcification at the surface or with-
in the interstitial spaces of matrices
formed. Additional calcification
inhibitors include phosvitin or other
phosphoproteins, dyes, such as alizarin
red 5, and methylene blue, calcium
chelators, such as EDTA and EGTA, and
other polyanions. The calcium inhibitor
chosen is preferably allowed to diffuse
freely into the tissueJ usually until
equilibrium is reached, which is after
about 12 hours.
3) To the solution containing the calcium
inhibitor and the tissue, an aliphatic
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diamine, preferably hexanediamine, is
added to provide additional binding
sites and cross-links in the subsequent
covalent binding steps. Although
diamines are preferable, other compounds
with free terminal amino or carboxyl
groups can be utilized. The diamine and
chondroitin sulfate may be added to the
solution at the same timeO but by adding
the calcium inhibitor first, more
polyelectrolytes are probably allowed to
diffuse into the tissue.
4) The tissues and additives are then
cross-linked by a water-soluble carbodi-
lS imide. Carbodiimides apparently form
peptide bonds by activation of carboxyl
groups to allow reaction with amino
groups. The cross-linking occurs at a
carbodiimide concentration of about 0.02
to about 0.1 molar~ preferably about
0.05 molar, in a balanced electrolyte
solution. The p~ should be between
about 4.7 and about 5.2, and is main-
tained at about 5.0 by the addition of
HCl. The preerred carbodiimide is 1-
ethyl-3-(3-dimethylaminopropyl)-car-
bodiimide ~Cl. If desired, ethanol and
other organic solvents may be added to
decrease the dielectric constant. The
cross-linking reaction is allowed to
~proceed from about 30 minutes up to 10
hours or moreO
5) After coupling is completed, the excess
reagents are removed by washing with a
~2~
balanced electrolyte solution at a
neutral pH, which also contains 0.05 M
EDTA~
.
6) The tissue is then transferred to a
neutral pH buffered solution, containing
about 0.2 to about 0.5 wt. % glutaralde-
hyde, preferably about 0.3 wt. %, in a
balanced electrolyte environment. This
final solution can be supplemented with
alcohol at a concentration of about 20
to about 50 wt. %, and surfactants, such
as anionic alkyl sulfates or alcohol and
Formaldehyde, for sterility and storage.
It is possible to modify the procedure
stated above~ For example, repeating the equili-
bration with the calcification inhibitor and subse-
quently reactivating the entire matrix with carbodi-
imide may be desirable.
Moreover, prior to or in conjunction with
the final glutaraldehyde treatment, anti-thrombogenic
compounds, such as heparin, which may also convey
additional attributes, may be added at a concentration
of about 0.2 to about l.0 wt. ~ Also, globular
proteins, small molecular weight peptides, or poly-
electrolytes, such as polylysine or polyglutamic acid,or mixed copolymers of poly-electrolytes, may be
added. By allowing these materials to diffuse into
the cross-linked matrix, further bridging between the
tissue components and exogenous materials may occur,
and the interstitial gaps may be filled. It is
believed that by filling the interstitial gaps the
deposit of calcium ions is minimized and hydroxy-
apatite and other crystal growth may be substantially
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inhibited.
Some basic improvements provided by the
present invention will now be discussed.
I. Immunogenicity
If material implanted in an organi~m can be
rendered insoluble, antigenicity can be substantially
eliminated~ To be recognized, antigens must be pre-
sented in a soluble form to activate the immune system
of the host organism. In many cases, materials that
are insoluble at the time of implantation can be
rendered soluble by naturally occurring enzymatic or
chemical processes. It is believed that the intro-
duction of sufficient cross-links prohibits the enzyme
systems of the host from solubilizing the implan~ed
material, ~hereby essentially eliminating antigen-
icity.
Glutaraldehyde treatment also introduces
cross-links, but for reasons not completely under-
stood, the cross-links generated in the present
invention render the entire device even less soluble.
Without being bound to any particular réason, perhaps
this reduced solubility is due to the presence of
cross-links different than those created when glutar-
aldehyde is used alone. Since glutaraldehyde
apparently acts primarily on lysine residues, the type
and amount of bridges produced are somewhat limited.
The present invention enhances the amount of cross-
linking by covalently attaching new amino groups to
~he structure, and additionally allows the use of
other moieties, such as peptide bound glutamic and
aspartic acids, to attach more cross-links in
different locations by the carbodiimide reaction
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mechanism.
II. Calcification
A significant, but often ignored, problem
associated with the implantation of grafts rich in
S collagen and elastin is the propensity of these grafts
to induce calcification. Collagen in particular has
an intrinsic ability to calcify, and a mixture of
collagen fibers with saturated solutions of calcium
and phosphate ions will induce nucleation, which is
closely followed by crystal growth. The addition of
polyanions, particularly sulfated polysaccharides, can
essentially prevent this nucleation process.
Some sulfated polysaccharides, such as
endogenous chondroitin sulfate, can be bound to the
collagen during the tanning procedure. But, these
p~lyanions are usually degraded by the host and sub-
sequently removed from the graft. Therefore, the
initial protection afforded to the tissue by these
materials is lost, and exposure of the functional
groups in collagen, as well as the new open spaces
generated, can now greatly enhance nucleation of
calcium and phosphate ions~ The process used in the
present invention covalently links these polyanions
to collagen, or some other primary structural compo-
nent of the prosthetic device, and sufficiently cross-
links the entire structure to prevent degradation and
crystal growth. The addition of any extraneous
calcification inhibitors that are also bound and
cross-linked can further minimize calcifîcationO
III. Host Induced Graft Destruction
Uncross-linked implanted, fresh heterografts
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or allografts are rapidly destroyed by the defense
mechanisms of a host organism. Adequate cross-
linking, which as previously discussed insolublizes
the tissue, can prevent this destruction. Again,
although glutaraldehyde induces a certain number of
cross-links, these have been shown to be inadequateO
Apparently, because of the different nature of the
cross-links produced in the present invention, greater
stabili~y can be obtained, while the actual density
of cross-links may be fewer. This is possible because
the cross-links have been designed to span a broader
set of distances, both inter- and intra-molecular; as
well as to join not only lysine residues present, but
also the free carboxyl groups of glutamic and aspartic
acid~ Apparently, these different types of cross-
` links give added resistance to the treated tissues
against enzymatic degradation, but importantly, with-
out significant decreases in the mechanical attributes
of the grafts.
20 IVo Compatibility With Blood Surfaces
Collagen, the primary structural component
of most animal tissues, is a well known platelet
aggregator and blood clot initiator. Since the
connective tissues used in prostheses are very rich in
collagen, the present invention utilizes substances
capable of reducing the tissues thrombogenic poten-
tial. Chondroitin sulfate also serves this purpose,
but additional compounds with antithrombogenic
properties, such as heparin, may be used. These
compounds, once covalently bound, sub tantially
decrease the ability of collagen to aggregate plate-
lets, thereby significantly decreasing the probability
of thrombus formation.
9~S~
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V. Changes in Mechanical Properties
The function of a transplanted device under
most circumstances will depend on the retention of
adequate visco-elastic behavior at a level particular-
ly suitable for the function that the graft is toperform. Maintaining the proper amount of elasticity
depends in-part on the degree of cross-linking.
Insufficient cross-links could allow for flow, enzyma-
tic de~radation, and subsequent destruction of the
physical integrity of the device. On the other hand,
too many cross-links can be conducive to brittleness,
and result in loss of function. The present invention
provides an adequate number of cross-links to help
retain the structural integrity of the implanted
device, but not so many or so clustered that elasti-
city is lost.
Al.TERNAT IVE EMBODI MENT
Tissues are received from the slaughter
house, cleaned to remove loosely adhering material,
and rinsed with cold phosphate-buffered ~ physiologi-
cal saline.
The tissues are then processed, usually at
about 4 degrees Centigrade, as follows:
1) treat with a glutaraldehyde solution at
a concentration between about 0.05 wt.
% and about 0.4 wt.-~, preferably about
0.15 wt. ~, for between about 12 and
about 64 hours, preferably about 48
hours;
2) rinse the tissue in phosphate buffered
5~
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saline to remove non-reacted glutaral-
dehyde;
3) place the tissue in a solution with a
pH of about 7.4 containing between
about 0.1 wt. ~ and about 2.0 wt. %
hexanediamine, preferably about 0.5
wt. %;
4) incubate for about 2 to about 10 hours~
preferably 4 hours;
5) transfer the tissues to a buffered
saline solution at a pH of about 4.9
that contains between about 0.1 and
about 1.0 w~. % of a water soluble
carbodiimide, prefereably 0.5 wt. %
1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide HCl:
6~ incubate for about 30 minutes to about
10 hours, preferably 1 hour, while
maintaining the pH of the entire
mixture at about pH 5.0 with an aqueous
HCl solution;
7) place the tissue in neutral, phosphate
buffered saline for rinsing from about
2 to about 12 hours, preferably about
6 hours;
8) place the tissue in a buffered neutral
saline solution that contains between
about 0.5 and about 3 wt. ~ of a sul-
phated polysaccharide, preferably about
1.0 wt. % chondroitin sulfate from
.
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whale and shark cartilage, the sodium
salt of mixed isomers (No. C-3129,
Sigma Chemical Company);
9) incubate for about 6 to about 16 hours,
preferably 12 hours, until equilibra-
tion (gentle mechanical shaking may be
used);
10) transfer the tissue to a buffered
saline solution~ at a pH of about 4.9
that contains about 0.1 to about 5 wt.
% of a water soluble carbodiimide,
preferahly about 0.5 wt. % of l-ethyl-
3 (3-dimethylaminopropyl)-carbodiimide
HCl, and about 0.1 to about 5 wt. % of
an aliphatic diamine, preferably about
0.5 wt. ~ hexanediamine;
11) incubate for about 30 minutes to about
10 hours, preferably 1 hour, while
maintaining the pH of the entire mix-
ture at about pH 5.0 with an aqueous
HCl solution;
12) rinse the tissue in a neutral, phos-
phate buffered saline solution from
about 2 to about 12 hours, preferably
about 6 hours;
13) transfer the tissue to a neutral, phos-
phate buffered saline solution contain-
ing between about 0.2 and about 2.0 wt D
~ of an antithrombogenic agent, prefer-
ably 1.0 wt~ % heparin;
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14) incubate for about 30 minutes to about
10 hours, preferably about 1 hour;
15) add, to the solution, glutaraldehyde to
a final concentration of between about
0.1 and about 1.0 wt. %, preferably
about 0.4 wt~ %;
16~ incubate for about 30 minutes to about
10 hours~ preferably about 1 hour;
17~ transfer to a final storage, neutral,
phosphate buffered solution containing
about 0O4 wt. % glutaraldehyde, prefer-
ably about 0.4 wt. ~, about 0.2 to
about 2.0 wt. % of formaldehyde, pre-
ferably about 1.0 wt. % and about 20 to
about 40 wt. % alcohol, preferably
about 30 wt. %.
Although the invention has been described in
detail, it will be understood by one of ordinary skill
in the art that various modifications can be made
without departing from the spirit and scope of the
invention. Accordingly, it is not intended that the
invention be limited except as by the appended claims.
