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Claims  |
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I claim:
1. A process for the treatment of collagenous tissue to adapt it for use in
a prosthetic implant and to promote the growth of endothelial cells
thereon after implantation, which comprises the steps of:
(a) contacting said tissue with at least one surfactant for a time
sufficient to substantially completely remove deleterious material and
open up the fibrous structure of the collagenous tissue;
(b) washing the resulting fibrous matrix to remove substantially all
surfactant;
(c) fixing the washed tissue with glutaraldehyde;
(d) treating the glutaraldehyde-fixed tissue with a
calcification-inhibiting agent, an agent which inhibits infiltration and
attack by phagocytic cells upon implantation and/or an agent which
inhibits infection; and
(e) treating the resulting agent/matrix tissue with a reducing agent to
stabilize the bonding of the glutaraldehyde of step (c) and the agent of
step (d) to the tissue.
2. A process according to claim 1, in which the collagenous tissue is
bovine or porcine pericardial tissue.
3. A process according to claim 1, in which the collagenous tissue is dura
mater, fascia lata, valve tissue or vascular graft tissue.
4. A process according to claim 1, in which the surfactant is in the form
of an aqueous solution containing 0.5 to 6% by weight of surfactant.
5. A process according to claim 4, in which said surfactant is a mixture of
an anionic surfactant and a non-ionic surfactant.
6. A process according to claim 5, in which the anionic surfactant is 1% by
weight sodium dodecyl sulfate and the non-ionic surfactant is 1% by weight
octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20)
sorbitan monooleate.
7. A process according to claim 6, in which the collagenous tissue is
contacted with said surfactant solution for about three hours at room
temperature.
8. A process according to claim 4, in which the surfactant is an amphoteric
surfactant.
9. A process according to claim 1, in which the collagenous tissue is
pre-washed with saline solution to remove excess blood and plasma
proteins, prior to treatment with surfactant.
10. A process according to claim 1, in which the fibrous matrix of tissue
resulting from the surfactant treatment is soaked in aqueous
glutaraldehyde solution for a time sufficient to fix the tissue by bonding
the glutaraldehyde molecules to substantially all the reactive amino
groups present in the protein molecules of the tissue.
11. A process according to claim 10, in which the concentration of
glutaraldehyde is 0.25 to 1% by weight.
12. A process according to claim 11, in which the tissue is soaked in 0.5%
by weight glutaraldehyde solution in the presence of 0.1M acetate buffer
for a period of about three and a half hours, after which excess
glutaraldehyde is washed from the tissue.
13. A process according to claim 10, in which the glutaraldehyde-fixed
tissue is treated with a calcification-inhibiting agent containing
reactive amino groups for a time sufficient to bond substantially all the
free reactive groups of the bonded glutaraldehyde molecules to the
reactive amino groups of the calcification-inhibiting agent.
14. The process according to claim 13, in which the
calcification-inhibiting agent is an amino diphosphonate.
15. A process according to claim 14, in which the amino diphosphonate is
selected from compounds of the formula:
##STR4##
16. A process according to claim 15, in which the amino diphosphonate is
3-amino-1-hydroxypropane-1,1-diphosphonic acid of formula (1) and the
tissue is soaked in fresh saturated solutions of said amino diphosphonate
in distilled water at a pH of 8.0 for three hours per day in each fresh
solution over a period of three days.
17. A process according to claim 16, in which the tissue is soaked in
glutaraldehyde between each fresh soaking with amino diphosphonate.
18. A process according to claim 14, in which the tissue is further treated
with 5 to 10 mg/ml of sodium borohydride to stabilize the bonding of the
amino diphosphonate and glutaraldehyde to the protein molecules of the
tissue.
19. A process according to claim 10, in which the glutaraldehyde-fixed
tissue is treated with (i) an agent which inhibits infiltration and attack
by phagocytic cells or (ii) an agent which inhibits infection.
20. A process according to claim 19, in which said agent (i) is a sporin
antibiotic having a free reactive amino group or methotrexate and said
agent (ii) is cephalosporin C.
21. A process according to claim 19, in which the tissue is treated with
sodium borohydride to stabilize the bonding of agent (i) or agent (ii) to
the treated tissue.
22. A process for the treatment of collagenous tissue to adapt it for use
in a prosthetic implant and to promote the growth of endothelial cells
thereon after implantation which comprises the sequential combination of
the following steps:
(1) contacting the tissue with at least one surfactant for a time
sufficient to substantially completely remove deleterious material and
open up the fibrous structure to form a matrix substantially free from
lipids, red blood cells, plasma protein, organelles, and dead cell
fragments;
(2) rinsing the cleaned fibrous matrix resulting from step 1 with distilled
water or saline solution to remove substantially all surfactant;
(3) soaking said matrix in aqueous glutaraldehyde solution for a time
sufficient to bond the glutaraldehyde molecules to substantially all the
reactive amino groups present in the protein molecules of the tissue;
(4) washing the glutaraldehyde-fixed tissue to remove excess
glutaraldehyde;
(5) treating the fixed tissue with an aqueous solution of amino
diphosphonate containing reactive amino groups for a time sufficient to
bond substantially all the free reactive groups of the bonded
glutaraldehyde molecules to the reactive amino groups of the amino
diphosphonate;
(6) washing to remove excess amino diphosphonate; and
(7) treating the diphosphonate-bonded tissue matrix with sodium borohydride
to stabilize the bonding of the amino diphosphonate and glutaraldehyde to
the protein molecules of the tissue; and
(8) washing to remove excess sodium borohydride and, if desired, storing
the resulting treated tissue in aqueous formaldehyde for subsequent use.
23. A process according to claim 22, in which the collogenous tissue is
bovine or porcine pericardial tissue.
24. A process according to claim 22, in which the collagenous tissue is
dura mater, fascia lata, valve tissue or vascular graft tissue.
25. A process according to claim 22, in which said collagenous tissue is
pre-washed with isotonic saline solution to remove excess blood and plasma
proteins prior to treatment with surfactant in step (1).
26. A process according to claim 22, in which step (1) is carried out with
an aqueous solution containing 0.5 to 6% by weight of surfactant.
27. A process according to claim 26, in which said surfactant is a mixture
of an anionic surfactant and a non-ionic surfactant.
28. A process according to claim 27, in which the anionic surfactant is 1%
by weight sodium dodecyl sulfate and the non-ionic surfactant is 1% by
weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene
(20) sorbitan monooleate.
29. A process according to claim 28, in which the collagenous tissue is
contacted with said surfactant solution for three hours at room
temperature.
30. A process according to claim 26, in which the surfactant is an
amphoteric surfactant.
31. A process according to claim 22, in which step (3) is conducted in a
solution having a glutaraldehyde concentration of 0.25 to 1% by weight.
32. A process according to claim 22, in which step (3) is conducted by
soaking the collagenous tissue in 0.5% by weight glutaraldehyde in the
presence of 0.1M acetate buffer for a period of about three and a half
hours.
33. A process according to claim 22, in which the amino diphosphonate used
in step (5) is selected from compounds of the formula:
##STR5##
34. A process according to claim 32, in which the amino diphosphonate is
3-amino-1-hydroxypropane-1,1-diphosphonic acid of formula (1) and the
tissue is soaked in fresh saturated solutions of said amino diphosphonate
in distilled water at a pH of 8.0 for three hours per day in each fresh
solution over a period of three days.
35. A process according to claim 34, in which the tissue is soaked with
glutaraldehyde between each fresh soaking with amino diphosphonate.
36. A process according to claim 22, in which step (7) is conducted with a
solution having a concentration of sodium borohydride of 5 to 10 mg/ml.
37. A fixed and stabilized collagenous tissue adapted for use in a
prosthetic implant produced by a process according to claim 22.
38. A storage-stable pack comprising a tissue according to claim 37
immersed in aqueous formaldehyde solution. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
This invention relates to a process for the treatment of collagenous tissue
to render it suitable for use in prosthetic implants, and to the resulting
tissue so treated. More particularly, the invention is concerned with a
process for the treatment of collagenous tissue to adapt it to be used in
a prosthetic implant and to promote the growth of endothelial cells
thereon.
Prosthetic implants for use in humans have been known for some time and it
also has been known to use natural tissue taken from animals including
humans. When natural tissue is used in an implant it is necessary to treat
it to avoid problems after implantation, for example excessive
mineralization or calcification and rejection by the body's immune system.
Numerous treatments for improving the stability of prosthetic devices made
from natural tissue have been proposed in the prior art.
Thus U.S. Pat. No. 4,378,224 issued Mar. 29, 1983 to Nimni et al discloses
a process for improving the biophysical stability of bioprostheses for
heterograft or allograft implantation made from animal tissue involving
the formation of cross-links in the protein structure of the tissue using
the known cross-linking agent glutaraldehyde and soaking the tissue in an
aqueous solution of a calcification inhibitor. Examples of suitable
calcification inhibitors mentioned by Nimmi et al are diphosphonates and
3-amino-1-hydroxypropane 1,1-diphosphonic acid is mentioned as a typical
diphosphonate, although no specific Example illustrating the use of this
compound is given by Nimni et al.
Furthermore, although Nimni et al refer to harvesting and cleaning of the
tissue prior to the glutaraldehyde treatment, there is no suggestion of
any pre-treatment with an appropriate surfactant to remove, substantially
completely, deleterious material present in the tissue. Accordingly, the
coating (column 2 line 23) provided by Nimni et al is essentially a
surface phenomenon and cross-linking with glutaraldehyde and stabilization
with the calcification inhibitor throughout the fibrous matrix of the
tissue is not and can not be achieved by the Nimni procedure.
U.S. Pat. No. 3,988,782 issued Nov. 2, 1976 to Dardik et al discloses the
preparation of prostheses in the form of tubes, patches and conduits from
arteries and veins of umbilical cords using glutaraldehyde as a hardening
agent.
U.S. Pat. No. 3,966,401 issued June 29, 1976 to Hancock et al discloses the
preparation of an implantable heart valve from porcine pericardial tissue
in which the tissue is treated with glutaraldehyde as a tanning agent.
The inhibitory effect of various diphosphonates on aortic and kidney
calcification in vivo is discussed in an article by M. Potokar and M.
Schmidt-Dunker appearing in Atherosclerosis, 30 (1978) 313-320.
U.S. Pat. No. 4,120,649 issued Oct. 17, 1978 to Schechter discloses the
treatment of transplants with glutaraldehyde to enhance the retention time
in the recipient. As in Nimni et al, supra, the treatment is essentially a
surface treatment and no additional stabilization or like treatment is
disclosed.
U.S. Pat. No. 3,562,820 issued Feb. 21, 1971 to Braun, discloses the
hardening with glutaraldehyde of tubular, strip and sheet form prostheses
based on biological tissue.
U.S. Pat. No. 4,098,571 issued July 4, 1978 to Miyata et al discloses a
process for preparing a heterograft substitute blood vessel which
comprises treating a pig blood vessel with a proteolytic emzyme to digest
unwanted material and retain collagenous and elastic fiber constituents
and then fixing the resulting blood vessel with, inter alia, a mixture of
formaldehyde and glutaraldehyde.
U.S. Pat. No. 4,323,358 issued Apr. 6, 1982 to Lentz et al discloses
treatment of a glutaraldehyde-fixed animal tissue with a solution of a
water-soluble salt of a sulfated higher aliphatic alcohol, such as sodium
dodecyl sulfate, allegedly to inhibit calcification of the tissue after
implantation.
Although all of the above prior art proposals have some degree of success,
for example by inhibiting calcification to some extent and improving the
biophysical stability of prosthetic implants to some extent, problems in
these areas still remain. Furthermore, none of the aforesaid prior art
disclosures express any recognition of the importance of promoting and
enhancing the growth of endothelial cells on the surfaces of prosthetic
implants.
The endothelium is a layer of flat cells lining various cavities within the
body, in particular blood vessels. The lining of endothelial cells
provides a smooth surface so that blood cells and platelets can flow
without being damaged. Endothelial cells are capable of producing and
secreting substances with a variety of actions and the actions occuring at
the blood-endothelial interface contribute towards the well-being of the
organism as a whole; for example, the intact endothelium is
nonthrombogenic because both circulating blood cells and the endothelial
surface have a negative charge and thus repel each other. Each endothelial
cell is closely linked to its adjacent cells and the endothelial layer
forms a selectively permeable membrane which resists the passive transfer
of the fluid and cellular phases of blood.
While the intact endothelium acts as a primary barrier against the leakage
of blood it also provides a prima facie indication to the body's immune
system that foreign materials are not present, at least outside the blood
vessels. However, if the endothelium is damaged, punctured or broken this
automatically induces a response by the immune system which defends
against foreign pathogens. The immune system, which is generally capable
of discriminating between self and foreign antigens, operates through a
complex assortment of lymphocytes and phagocytic cells whose activities
are adapted to produce a coordinated protective response to foreign
pathogens. Thus, among the phagocytic cells involved in the immune system,
white blood cells or leukocytes function primarily to defend the body
against microorganisms. Another important group of phagocytes is the
macrophages which are widely distributed throughout the body and act in
concert with other phagocytes associated with the linings of blood
vessels, i.e. the endothelium, in, for example, the bone marrow, liver,
spleen and lymph nodes.
A more detailed description of the immune system is not considered
necessary for a full understanding of the present invention, but
recognition of the role played by endothelial cells is important for an
appreciation of the improvement provided by the invention over the prior
art.
Surprisingly, it has now been found that by performing the process of the
present invention and, in particular, ensuring substantially complete
removal of deleterious material from collagenous material by the essential
initial step of said process, the in vivo growth of endothelial cells upon
prosthetic implants formed from tissue treated by the invention process is
promoted. In addition to a marked improvement in the inhibition of
mineralization or calcification upon implantation, this permits the
formation of implants which are less susceptible to rejection by the
body's immune system than any produced by prior art procedures.
SUMMARY OF THE INVENTION
In accordance with the invention there is provided a process for the
treatment of collagenous tissue to adapt it for use in a prosthetic
implant and to promote the growth of endothelial cells thereon after
implantation, which comprises the steps of:
(a) contacting said tissue with at least one surfactant for a time
sufficient to substantially completely remove deleterious material and
open up the fibrous structure of the collagenous tissue;
(b) washing the resulting fibrous matrix to remove substantially are
surfactant;
(c) fixing with glutaraldehyde; and
(d) treating with a calcification-inhibiting agent, an agent which inhibits
infiltration and attack by phagocytic cells upon implantation and/or an
agent which inhibits infection; and, if desired,
(e) treating the resulting agent/matrix tissue with a bond-stabilizing
agent.
Collagen is a fibrous protein which occurs in vertebrates as the primary
constituent of connective tissue fibrils. There are seven different types
of collagen and type I is generally used for implants. Fibrous animal
tissue normally contains collagen in association with other proteinaeous
material, particularly elastin. As used herein the term collagenous tissue
is intended to mean collagen tissue, particularly type I collagen,
mixtures of collagen and elastin and animal tissues containing a
significant proportion of collagen with or without elastin or other
proteinaceous material. An essential requirement of the collagenous tissue
to be used in the invention is that the protein molecules thereof contain
free amino groups adapted to react with fixing or tanning reagents such as
glutaraldehyde.
The preferred collagenous tissue is bovine pericardial tissue or porcine
pericardial tissue. Such tissue is particularly suitable for the formation
of the tissue leaflets in prosthetic heart valves, particularly those made
in accordance with the teachings of Ionescu et al U.S. Pat. No. 4,388,735
issued June 21, 1983.
Other suitable forms of collagenous tissue which may be treated by the
process of the invention are dura mater, fascia lata, valve tissue and
vascular graft tissue.
Collagenous tissue, for example pericardial tissue, as it is initially
removed from an animal requires cleaning to free it from unwanted
contaminants. Usually the tissue is washed with sterile isotonic saline
solution to remove excess blood and plasma proteins, and this conventional
pre-washing is a desirable initial step before performing the essential
step according to the process of the invention of contacting tissue with
at least one surfactant for a time sufficient to substantially completely
remove deleterious material.
As used herein the term deleterious material is intended to mean material
which blocks or clogs the fibrous matrix of the collagen or
collagen/elastin tissue to be used as a prosthetic implant, which
material, if not removed, would provide sites to initiate an immune
response in the host organism with consequential rejection of the implant
or at least attack by host phagocytes. Deleterious material includes
lipids, including lipo-protein and phospholipids, red blood cells, plasma
protein, organelles and dead cell fragments, as well as free fatty acids,
cholesterol, cholesterol esters and triglycerides.
The removal of deleterious material by the surfactant-treating step of the
invention opens up the fibrous structure of the collagenous tissue and
this enables the glutaraldehyde used in the subsequent fixing step to
subtantially completely infiltrate the fibrous matrix of the collagenous
tissue so that the reactive groups on the glutaraldehyde molecules bond to
the free amino groups on the protein molecules of the collagenous tissue
throughout the matrix.
Thus the surfactant treatment of the present invention serves the double
purpose of, firstly, enabling the fibrous matrix of collagenous tissue to
be thoroughly fixed throughout the matrix rather than merely on the
surface; and, secondly, deleterious material is removed from the
interstices of the fibrous matrix and is no longer present to be entrapped
below the surface by the subsequent fixing step and to be available to
present problems of rejection or attack by host phagocytes upon
implantation. Prior art procedures which have advocated treatment with
surfactants after the fixing step, for example U.S. Pat. No. 4,323,358
supra, are substantially ineffective for removing deleterious material
which is effectively bonded to the tissue by the fixing agent.
It has been found that the particular sequence of steps according to the
present invention provides a significant improvement in terms of implant
retention over the prior art.
The surfactant used in the surfactant-treating step of the invention is a
potent agent for removing deleterious material from animal tissue and care
must be taken not to overdo the cleaning action and thereby damage the
base tissue by using too strong a solution. On the other hand, the
concentration of surfactant and the period of treatment must be sufficient
to achieve the desired result of substantially completely removing the
deleterious material. Within these criteria it is preferred to use the
surfactant in the form of an aqueous solution containing 0.5 to 6% by
weight of surfactant. A suitable treatment time is from two to six hours,
preferably about three hours.
The surfactant may be an anionic surfactant, a non-ionic surfactant, an
amphoteric surfactant or a mixture thereof.
Examples of suitable anionic surfactants are sodium dodecyl sulfate, sodium
dodecyl sulfoacetate and sodium salt of alkaryl polyether sulfonate.
Examples of suitable non-ionic surfactants are octylphenoxy polyethoxy
ethanol (Triton X-100), polyoxyethylene (20) sorbitan monooleate (Tween
80), polyoxyethylene (20) sorbitan monostearate (Tween 60). Examples of
suitable amphoteric surfactants are sulfobetaines commonly known as
Zwittergents.
It has been found that particularly advantageous results are obtained if
the surfactant is mixture of an anionic surfactant and a non-ionic
surfactant; and a particularly preferred surfactant solution is one in
which the anionic surfactant is 1% by weight sodium dodecyl sulfate and
the non-ionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol
and/or 1% by weight polyoxyethylene (20) sorbitan monooleate. Preferably
the collagenous tissue is contacted with said surfactant solution for
about three hours at room temperature.
The surfactant is not only a potent cleansing agent but also a potential
toxin and, accordingly, it is an important feature of the invention that,
after the surfactant treatment, the fibrous matrix of collagenous tissue
is thoroughly washed to remove substantially all surfactant. This washing
step may be conducted in any conventional manner, for example with saline
solution or distilled water, and, to ensure substantially complete removal
of surfactant, the washing is continued until the formation of bubbles
ceases.
After the above-described treatment with surfactant and the washing step to
remove substantially all trace of surfactant the fibrous matrix of tissue
resulting from the surfactant treatment is soaked in aqueous
glutaraldehyde solution for a time sufficient to fix the tissue by bonding
the glutaraldehyde molecules to substantially all the reactive amino
groups present in the protein molecules of the tissue. A suitable time for
the fixation step is from two to twelve hours and substantially complete
fixation is achieved preferably by the repeated soaking procedure
described hereinafter. Preferably, the concentration of glutaraldehyde is
0.25 to 1% by weight.
Fixation of animal tissue with glutaraldehyde to improve its
characteristics and render it adaptable for prosthetic implants is known
in the art and this step, in and of itself, is not claimed to be
inventive. However, the special contribution provided by the invention
with regard to this step is two-fold:
Firstly, the soaking with glutaraldehyde is carried out only after
deleterious material has been substantially completely removed from the
collagenous tissue by the surfactant treatment; thus ensuring that the
fibrous matrix is adapted to be fixed throughout, rather than merely on
its surfaces.
Secondly, substantially complete fixation throughout the fibrous matrix is
ensured by soaking the tissue in the glutaraldehyde for a time sufficient
to bond the reactive groups on the glutaraldehyde molecules to
substantially all the reactive amino groups present in the protein
molecules of the tissue.
The described result is preferably achieved by repeated soakings in
glutaraldehyde to effect multiple cross-linking in accordance with the
procedure described hereinafter. The need for repeated soakings, not only
in glutaraldehyde to effect multiple cross-linking, but also in
calcification-inhibiting agents and anti-phagocytic agents, as described
hereinafter, to achieve the cumulative saturation effect provided by the
process of this invention has not been achieved in the prior art.
According to a preferred embodiment of the invention the tissue is soaked
in 0.5% by weight glutaraldehyde solution in the presence of 0.1M acetate
buffer for a period of about three and a half hours. Subsequently, excess
glutaraldehyde is washed from the tissue.
An important aspect of the invention is the inhibition of calcification on
prosthetic implants formed from tissue treated in accordance with the
process of the invention.
It has been found that the presence of phosphate ions tends to increase the
occurrence of calcification and accordingly the use of phosphate buffers
in the steps of the inventive process is to be avoided, notwithstanding
the efficiency of the intermediate washing steps.
Since control of pH is an important feature during the process, such
control preferably is achieved with a non-phosphate buffer, preferably an
acetate buffer.
The tissue fixed with glutaraldehyde is further treated in accordance with
the invention firstly with a calcification-inhibiting agent and/or an
agent which inhibits infiltration and attack by phagocytic cells upon
implantation and finally with an agent, usually a reducing agent, which
stabilizes the molecular bonds of the resulting agent/matrix tissue.
Mineralization, or more particularly calcification, on and around tissue
implants after implantation results in reduced flexibility of the tissue
and therefore decreased efficiency in the operation of the prosthesis in
the host body. Various treatments have been proposed in the prior art to
inhibit or reduce calcification and these have met with some degree of
success. In particular, the use of specific compounds to inhibit
calcification is known in the art. However, the special application of
known calcification inhibitors, especially amino diphosphonates, in
accordance with the process of the present invention results in a
substantial improvement over prior art treatments and unexpected
advantages in areas not investigated in prior art procedures.
Thus, prosthetic implants made from collagenous tissue treated in
accordance with the process of the present invention are found to be
effective in resisting not only calcification but also thrombosis,
infection and degeneration. These advantageous characteristics, which are
exhibited to a degree substantially greater than that achieved in the
prior art, are attributable to the fact that endothelial cell coverage on
the implant is encouraged and the promotion of such coverage protects the
implant from the reactions leading to thrombosis, calcification, infection
and degeneration.
The stated improvement is attained by the particular combination of steps
described above in which the agent used in the further treatment of the
fixed tissue, as well as a calcification agent, may be an agent which
inhibits infiltration and attack by phagocytic cells, for example, a
sporin antibiotic having a free reactive amino group or methotrexate; or
an agent which inhibits infection, preferably cephalosporin C.
The said sporin antibiotic is derived from cyclosporin A, a known
immunosuppresive drug having a molecular weight of 1202 and the structural
formula illustrated in FIG. 7 of the accompanying drawings.
For use as a treating agent in the process of the present invention the
cyclopsorin A ring is opened at the position indicated by the arrow in the
formula, to provide a free amino group for reaction with the free reactive
groups on the glutaraldehyde molecules attached to the fixed tissue.
Methotrexate, or
N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic
acid is a known folic acid antagonist and antimetabolite having the
formula:
##STR1##
This drug has two free amino groups available for reaction with the
reactive groups on the glutaraldehyde-fixed tissue matrix.
Cephalosporin C is a known potent inhibitor of infection.
For convenience in terminology, the calcification inhibiting agents and the
immuno-suppressive agents and drugs used in the post-fixing step of the
process according to the invention are referred to hereinafter by the
generic term "drug" and, under this terminology, the desired effect
produced by the process may be termed "drug immobilization" or
"immunosuppression".
One of the advantageous results achieved by the drug immobilization
provided by the process of the invention is an effective balance between:
(1) the encouragement or promotion of endothelial cell coverage;
(2) the enhancement of would healing; and
(3) inhibition of rejection and attack by macrophage and other phagocytes.
A preferred application for tissue subjected to drug immobilization by the
process of the invention is in the production of prosthetic heart valves
wherein the leaflets and sewing ring are formed from the treated tissue. A
particularly preferred embodiment of the tissue is produced when the drug
is an amino diphosphonate calcium inhibitor and it has been found that
prosthetic heart valves made from the preferred embodiment conform to the
above balance in that examination of the prosthesis some two months after
implantation shows:
(1) Substantially complete endothelial cell coverage over the leaflets and
sewing ring and no evidence of thrombosis, calcification, infection or
degeneration;
(2) Substantially complete would healing with no evidence of macrophage
debris or macrophage factor; and
(3) No evidence of rejection or inhibition of endothelial cell growth by
microphage (monocyte) attack.
The drug immobilization process preferably is completed by stabilizing the
fixed and drug-treated tissue with a reducing agent. Although in theory
any reducing agent which will effectively reduce double bonds between
carbon and nitrogen atoms may be used for this step including
cyanoborohydride; to avoid any possible problems from toxic residues, the
preferred reducing agent is sodium borohydride (NaBH.sub.4).
Thus, the preferred embodiment of the invention provides a process for the
treatment of collagenous tissue to adapt it for use in a prosthetic
implant and to promote the growth of endothelial cells thereon after
implantation which comprises the sequential combination of the following
steps:
(1) contacting the tissue with at least one surfactant for a time
sufficient to substantially completely remove deleterious material and
open up the fibrous structure to form a matrix substantially free from
lipids, red blood cells, plasma protein, organelles, and dead cell
fragments;
(2) rinsing the cleaned fibrous matrix resulting from step 1 with distilled
water or saline solution to remove substantially all surfactant;
(3) soaking said matrix in aqueous glutaraldehyde solution for a time
sufficient to bond the glutaraldehyde molecules to substantially all the
reactive amino groups present in the protein molecules of the tissue;
(4) washing the glutaraldehyde-fixed tissue to remove excess
glutaraldehyde;
(5) treating the fixed tissue with an aqueous solution of amino
diphosphonate for a time sufficient to bond substantially all the free
reactive groups of the bonded glutaraldehyde molecules to the reactive
amino groups of the amino diphosphonate;
(6) washing to remove excess amino diphosphonate; and, if desired,
(7) treating the diphosphonate-bonded tissue matrix with sodium borohydride
to stabilize the bonding of the amino diphosphonate and glutaraldehyde to
the protein molecules of the tissue;
(8) washing to remove excess sodium borohydride and, if desired, storing
the resulting treated tissue in aqueous formaldehyde for subsequent use.
The preferred collagenous tissue is bovine or porcine pericardial tissue.
Alternatively, the collagenous tissue may be dura meta, fascia lata, falve
tissue or vascular graft tissue.
Preferably, the collagenous tissue is pre-washed with isotonic saline
solution to remove excess blood and plasma proteins prior to treatment
with surfactant in step (1).
Preferably, step (1) is carried out with an aqueous solution containing 0.5
to 6% by weight of surfactant; the surfactant preferably being selected
from those listed above. Particularly desirable results are obtained when
said surfactant is a mixture of an anionic surfactant and a non-ionic
surfactant.
A particularly preferred surfactant solution is one in which the anionic
surfactant is 1% by weight sodium dodecyl sulfate and the non-ionic
surfactant is 1% by weight octylphenoxy polyethoxy ethanol and /or 1% by
weight polyoxyethylene (20) sorbitan monooleate.
In carrying out step (1) it has been found that the sufficient time
requirement is fulfilled when the collagenous tissue is contacted with
said surfactant solution for two to six hours, preferably about three
hours, at room temperature.
The fixing treatment of step (3) preferably is conducted in a solution
having a glutaraldehyde concentration of 0.25 to 1% by weight.
As described above, when treating collagenous tissue, particularly
pericardial tissue, with a fixing solution this step is conducted by
soaking the collagenous tissue in 0.5% by weight glutaraldehyde in the
presence of 0.1M acetate buffer for a period of about three and a half
hours.
The glutaraldehyde-fixed tissue is then carefully washed, for example with
deionized or acetate-buffered water, to remove excess glutaraldehyde and
then treated according to step (5).
Preferably, the amino diphosphonate used in step (5) is selected from
compounds of the formula:
##STR2##
A particularly preferred amino diphosphonate is
3-amino-1-hydroxypropane-1,1-diphosphonic acid of formula (1) and
preferably the tissue is soaked in fresh saturated solutions of said amino
diphosphonate in distilled water (16 mg/ml.) at a pH of 8.0 for three
hours per day in each fresh solution over a period of three days.
Particularly advantageous results are obtained if the tissue is soaked in
glutaraldehyde between each fresh soaking in amino diphosphonate. This in
effect means repetition of steps (3), (4) and (5).
The cumulative effect of multiple cross-linking on drug uptake (i.e. amino
diphosphonate uptake) by following this procedure is illustrated
graphically in FIG. 3 of the accompanying drawings; and this effect, as
well as the effect of surfactant, temperature and fixation time on drug
uptake is discussed hereinafter.
The next preferred step in the treatment of the tissue after drug uptake is
stabilization with sodium borohydride and this step (7) preferably is
conducted with a solution having a concentration of sodium borohydride of
5 to 10 mg/ml.
The process of the preferred embodiment is summarized in the following
reaction scheme, wherein protein --NH.sub.2 represent a molecule of
collagen or elastin in the collagenous tissue containing one free amino
group and DP-NH.sub.2 represent a molecule of amino diphosphonate
containing one free amino group.
##STR3##
In the above reaction scheme the numerals (3), (5) and (7) identify the
relevant steps of the process and the final formula illustrates a fully
saturated conjugate containing a terminal diphosphonate group.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be more particularly described by reference to
the accompanying drawings in which:
FIG. 1 is a graph illustrating the effect of temperature and surfactant on
drug uptake;
FIG. 2 is a graph illustrating the effect of fixation time and temperature
on drug uptake;
FIG. 3 is a graph illustrating the effect of multiple cross-linking
(fixation) on drug uptake;
FIG. 4 is a graph illustrating the effect of pH on drug bonding;
FIG. 5 is a graph illustrating the enhanced drug binding to pericardium
achieved following the initial treatment with surfactant;
FIG. 6 is a scanning electron micrograph, magnification X2000, showing
endothelial cell coverage on a valve made from pericardial tissue treated
in accordance with the invention after two months in a calf; and
FIG. 7 illustrates the structural formula of cyclosporin A.
In the graphs of FIGS. 1 to 5, the term "drug" means
3-amino-1-hydroxypropane-1, 1-diphosphonic acid. Comparable results are
obtainable with other amino diphosphonates in accordance with the
invention.
The following Example illustrates in more detail the preferred embodiment
of the invention.
EXAMPLE
The pericardium was removed from the heart of a calf. The pericardial
tissue was then washed with 0.9% saline solution to remove excess blood
and plasma proteins.
Fatty tissue and thick adherent tissue were removed.
The cleaned fat-free pericardial tissue was then cut into (5-10
cm.times.5-10 cm) pieces and each piece of tissue (hereinafter referred to
simply as "tissue") was treated according to the following procedure.
The tissue was immersed in a surfactant solution comprising 1% by weight
sodium dodecyl sulfate and 1% by weight octylphenoxy polyethoxy ethanol,
commercially available under the Trade Mark Triton X-100. The tissue was
soaked in the surfactant solution at room temperature (23.degree. to
25.degree. C.) for a period of three hours.
The tissue was removed from the surfactant solution and thoroughly rinsed
with saline solution in a strainer until no more bubbles were seen coming
from the tissue and vesicles were removed by suction and washings. It is
to be understood that the importance of this washing step is to ensure
substantially complete removal of surfactant from the tissue and the
nature of the washing solution is not critical, for example, distilled
water, deionized water or 0.05M acetate buffer solution having a pH of 5.5
may be used instead of saline solution.
After the aforesaid washing step, the tissue was soaked in 0.5% by weight
glutaraldehyde in 0.1M acetate buffer solution for about three and a half
hours.
The fixed tissue was rinsed in 0.05M acetate buffer (or deionized water) to
remove excess glutaraldehyde and immersed in a saturated drug solution
comprising 16 mg/ml. of 3-amino-1-hydroxypropane-1,1-diphosphonic acid in
0.05M acetate buffer. The tissue was soaked in the drug solution for a
period of two to three hours.
After the initial drug bonding step the tissue was reimmersed in 0.05M
acetate buffer/glutaraldehyde solution and soaked therein for twelve
hours.
The tissue was then again rinsed and soaked in drug solution for a period
of two to three hours.
The fixation and drug-bonding steps were then repeated two more times.
The cumulative effect of repeating the glutaraldehyde and amino
diphosphonate treatments is illustrated graphically in FIG. 3 of the
accompanying drawings and these steps may be repeated as long as there is
any significant increase in drug uptake. In practice, the performance of
each step four times, i.e. three repetitions of each, is normally
sufficient to obtain substantially maximum uptake of drug. The overall
process is dependent upon the amino group conjugation of amino
diphosphonate via glutaraldehyde to the amino acid (lysine) of the
collagen or elastin in the tissue.
After the final repetition of the drug treatment the tissue was soaked in a
solution containing 5 mg/ml of sodium borohydride for thirty minutes at
25.degree. C.
Finally the tissue was removed from the sodium borohydride solution, rinsed
to remove excess borohydride and placed in storage under 0.5%
glutaraldehyde or formaldehyde solution until required for use.
Tissue valves prepared from tissue treated according to the above procedure | | |
