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FIELD OF THE INVENTION
This invention relates to xenogeneic tissue implantation in human tissue
repair and prostheses.
BACKGROUND OF THE INVENTION
Implantation in humans of xenogeneic tissue, i.e. tissue from a species
other than human, has been carried on extensively for more than two
decades. Xenogeneic implants are useful in replacing human tissues which
are damaged by pathological or traumatic injury. Such implants have been
used for replacing heart valves, ligaments, tendons and skin, for example.
Many techniques for preparation and treatment of xenogeneic tissue have
been developed for many types of prosthetic and tissue repair applications
in the human body. For example, treatment of such tissue with collagen in
various forms and degrees of denaturization are known (U.S. Pat. Nos.
3,563,228, Seiderman, 3,949,073, Daniels et al, and 4,233,360, Luck, et
al.) Treatment of graft tissues with aldehydes, and glutaraldehyde in
particular, is well known (see, for example, U.S. Pat. No. 3,988,872,
Dardik, et al, which is but one of many disclosures of the use of
glutaraldehyde in tissue treatment.)
Exemplary of the state of the art are the following U.S. Pat. Nos.: Angell
et al., U.S. Pat. Nos. 4,035,848 and 4,247,292 and Hancock, et al, U.S.
Pat. No. 4,050,893--glutaraldehyde treatment of porcine heart valves;
Schechter, U.S. Pat. No. 4,120,649--glutaraldehyde treatment of pigskin,
human tissue, and amniotic membranes; Holman, et al, U.S. Pat. Nos.
4,239,492 and 4,240,794--glutaraldehyde treatment of umbilical cord tissue
for vascular grafts; Ketharanathan, U.S. Pat. No.
4,319,363--glutaraldehyde treatment of artificially induced tubular
structure of collagenous tissue; Lentz et al, U.S. Pat. No.
4,323,358--treatment of implant tissue with glutaraldehyde and wetting
agent; Wright, U.S. Pat. No. 4,350,492, and Lane, U.S. Pat. Nos. 4,372,743
and 4,443,895--heart valve prosthesis from glutaraldehyde treated porcine
heart valve; Kurland, U.S. Pat. No. 4,400,833--tendons and ligaments from
cows and pericardium or other porcine tissue treated with glutaraldehyde
and reinforced with synthetic mesh structure; Pollock, et al, U.S. Pat.
No. 4,402,697--treatment of implant tissue with phosphate ester and
glutaraldehyde; and Pollock, U.S. Pat. No. 4,405,327--treatment of implant
tissue with quaternary ammonium compounds and glutaraldehyde.
One of the major problems which have had to be overcome in the preparation
of implant tissues is the histocompatibility barriers which the human
recipient erect when a non-self material is introduced. Immune rejection
of transplants has been and remains a concern, even though much work has
been done in this area of medical-immunochemical technology.
Prevention of or inhibition of infection is another goal in the field of
implantation.
As illustrated by the previously cited prior art, glutaraldehyde has been
reported as being effective in reducing antigenicity and inhibiting
infection of implant tissue. Glutaraldehyde cross-links proteins rapidly
and effectively, and causes the cross-linking of proteins in the tissue
being treated. This treatment increases resistance to proteolytic cleavage
and hence increases resistance to enzymatic degradation. The treatment of
implant tissue with glutaraldehyde is sometimes referred to as "tanning"
because it crosslinks the protein and inhibits enzymatic and biochemical
degradation of the tissue, comparable in general to the effect of tanning
leather. Glutaraldehyde is also often used as the preservative in aqueous
solution for storing tissues after treatment.
Xenografts prepared by the prior art methods suffer from three principal
disadvantages, none of which precludes their use for human implantation,
but which, nevertheless, represent deterrents to their greater
acceptability for use in human tissue replacement.
First, despite the fact that their primary constituent is only weakly
immunogenic, by virtue of collagen being present in all mammalian species,
and that the crosslinking process further reduces their immunogenicity,
xenografts are capable of stimulating the formation of circulating
antibodies in the human system, indicating some residual immunogenicity.
Second, the use of some chemical sterilants increases the risk that toxic
response will be encountered in sensitive individuals, even after thorough
rinsing of the xenograft prior to implantation.
Third, crosslinked xenografts are somewhat stiffer than the native tissue
and this stiffness, or lessened compliance, is undesirable, since the
xenograft functions best when it preserves the original biomechanical
properties that nature intended for the function of that particular
tissue.
A feature of the present invention is that it encompasses methods of
radiating pre-crosslinked tissue which reduces, or eliminates, one or all
of these three disadvantages.
Collagenous tissue in uncrosslinked form has been reported to be seriously
degraded by radiation. Apparently, this reported result has deterred
investigators from studying all aspects of radiation treatment of
xenogeneic tissue.
SUMMARY OF THE INVENTION
The present invention contemplates, as an article of commerce for use in
implantation therapy, a tissue which has been treated to crosslink the
proteins, i.e. a cross-linked tissue, and which, thereafter, has been
subjected to radiation sufficient to effect sterilization and to reduce
immunogenicity and increase compliance, but insufficient to cause
significant degradation.
As a method, the present invention comprises the steps of crosslinking
proteins in a tissue and thereafter sterilizing the tissue with radiation
in an amount sufficient to effect sterilization and to reduce
immunogenicity and increase compliance, but insufficient to cause
significant degradation.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The method of the present invention is applicable to most and probably all
xenogeneic tissue preparation. Examples of the types of xenogeneic tissues
which can be prepared by and which results from the present invention
include tendons, ligaments, pericardial membrane, skin, umbilical cord
membrane, heart valve tissue, vascular tissue and the like.
The first step in the process of preparing the tissues of this invention is
to effect crosslinking of the protein in the tissue. Any crosslinking
reagent may be used, glutaraldehyde currently being one of the preferred
reagents. Other aldehydes, however, or other crosslinking materials may be
used.
The crosslinking can be carried out in any desired method. Many such
methods are described in the prior art. Generally, the crosslinking step
comprises soaking the tissue in glutaraldehyde solution, or other aldehyde
containing solution, for from a few minutes to several days, depending
upon the rate of crosslinking reaction. The rate of crosslinking reaction
can be controlled by controlling the concentration of glutaraldehyde and,
to a lesser extent, by controlling the pH and/or the temperature of the
crosslinking reagent. The concentration of the glutaraldehyde is typically
from about 0.1% to 5.0% The solution is typically buffered to about pH 7
to 9 with any suitable buffer, e.g. conventional bicarbonate, citrate, and
phosphate buffers and the like. Time and concentration are, of course,
related and considerable variation in both are well known in the art. The
solution may include one or a number of crosslinking materials, such as,
for example, formaldehyde, glyoxal, and/or dialdehyde starch. This step
is, of course, well known and reference may be made to any number of prior
art patents and publications for guidance as to this step. For example,
one well known treatment method for crosslinking tissue, i.e. crosslinking
the proteins in the tissue, is described by Yarbrough, et al; Structural
alterations in tissue cardiac valves implanted in patients and in calves.,
J Thoracic and Cardiovascular Surgery, March 1973, pp. 364-74.
The second step of the present process is to irradiate the tissue which has
been previously crosslinked as described. It is, of course, well known
that sterilization can be accomplished by radiation with high energy
photons such as X-rays and gamma rays; however, it is also known that
proteins generally and collagen in particular is seriously degraded by
such radiation. One would not, therefore, normally consider radiation
sterilization for xenogeneic tissue implants.
An optional step may be carried out before the second step just described.
In some instances, a chemical sterilization or partial sterilization may
be carried out to reduce the bioburden before the irradiation step is
performed.
The irradiation step of this invention can be carried out using very high
energy X-radiation; however, it is considered preferable and easier to
irradiate the pre-crosslinked xenogeneic tissue with gamma radiation such
as, for example, from a conventional Co.sup.60 gamma source.
The amount of radiation is not critical in that some variation is possible,
though it is possible to over-irradiate the tissue. In practice, the
amount of radiation which first begins significantly to degrade the
xenogeneic tissue in which the protein has been substantially crosslinked
is determined. This is the upper level of radiation normally employed. The
amount of radiation which is just sufficient to sterilize the xenogenic
tissue in which the protein has been substantially crosslinked is
determined, thus establishing the minimum level of radiation. The
preferred range of irradiation is from at least the level or irradiation
which is reliable to effect sterilization. In most instances, is desirable
to effect two or more, and preferably about two or about five, times the
minimum quantity of radiation necessary to effect sterilization, but less
than the amount of irradiation which begins significantly to degrade the
previously crosslinked xenogeneic tissue. The preferred range of radiation
using cobalt-60 gamma radiation is from about two to about eight megarads,
usually in the range of from two to five megarads. There are some
indications that it is preferable to utilize lower irradiation flux for
longer periods of time, as compared with a higher irradiation flux for a
shorter period of time, to effect the same quantity of irradiation;
however, this phenomenon has not been fully explored.
Radiation is carried out in the conventional manner, i.e. by placing the
xenogeneic tissue in which the protein has been substantially crosslinked
in a suitable glass or other container, and placing the container adjacent
the radiation source and opening a path or slit between the radiation
source and the tissue to generally uniformly expose the tissue in the
gamma rays emitted by the radioactive decay of Co.sup.60, or such other
gamma ray source, or an equivalent high energy radiation source such as
may, for example, result from electron beam acceleration, as may be
available.
Effective sterilization is easily determined using conventional
microbiologial techniques, such as the inclusion of suitable biological
indicators in the radiation batch, as is now conventional, or the older
but suitable method of contacting the tissue with a culture medium and
incubating the medium to determine sterility of the tissue. These are, of
course, textbook methods.
Degradation of the pre-crosslinked xenogeneic tissue by irradiation is also
determined using well known and conventional tests and criteria, i.e.
reduction in shrink temperature, T.sub.s ; susceptibility to enzyme
attack, e.g. collagenase; extractability of degradation products, e.g.
collagen fragments; and decrease in physical properties such as tensile
strength.
As expected, radiation sterilization was effective in obviating the need
for toxic sterilizing chemicals. Contrary to all expectations, however,
the amount of radiation required for sterilization did not degrade the
xenogeneic tissue in which the protein has been substantially crosslinked.
Surprisingly, the physical characteristics of the irradiated xenogeneic
tissue in which the protein had previously been substantially crosslinked
were greatly improved. Tensile strength remain approximately as in the
unirradiated tissue, but the irradiated tissue was less rigid, more
flexible and compliant and, therefore, superior to the unirradiated tissue
for most implant purposes, being much more like the original tissue than
the unirradiated tissue.
Another surprising discovery was also made. One would expect that one of
the effects of irradiation would be to break some of the crosslinked bonds
which had been previously effected in the tissue and, therefore, to expose
sites susceptible to enzyme attack and increase the potential antigenicity
of the tissue. The contrary was found, however. The antigenicity of the
irradiated tissue was reduced. The reduced antigenicity of the irradiated
xenogeneic tissue in which the protein had previously been substantially
crosslinked was shown by comparing reactivity against collagen-induced
antibodies of both animal and human origin.
It is presumed that some crosslinking is broken by the irradiation, but
apparently not the crosslinks which would expose sites for enzyme attack
or antigenic determinant sites; however, the precise change which occurs
is unknown and unexplainable by an hypothesis of which the inventor is
aware, as the results are most unexpected and run contrary to the
conventional wisdom of the crosslinked tissue art.
Treatment of bovine tendon is given to exemplify the invention, for, as
previously explained, the source of nature of the tissue is not critical
and virtually any tissue may be treated and used according to this
invention. Any suitable bovine tendon is cleaned, excess tissue, fat, etc.
is removed and, generally, is prepared in the manner in which xenografts
are conventionally prepared. The bovine tendon tissue is then
pre-crosslinked, either free-floating or in a fixed configuration as
desired, in glutaraldehyde, or other crosslinking reagent, as described
above and in the prior art, e.g. as described by Yarbrough, et al, supra.
The pre-crosslinked bovine tendon thus prepared is placed in sterile
physiological buffered saline solution in a glass or other container and
the container is exposed to from two or five magarads of sterilizing high
energy radiation, typically gamma radiation from Co.sup.60. Sterilization
is assured by appropriate control or testing, and the tissue is checked to
assure that no significant degradation has occurred, using the methods
described. The container may then be stored indefinitely and, when used,
need only be removed from the container and implanted.
As another example, by way of illustration, and not of limitation, fresh
bovine or porcine diaphragm tissue is received from the slaughter house,
inspected to meet vendor specifications, and thoroughly rinsed in pH 7.4
phosphate buffered solution. The diaphragm tissue is dissected, separating
and discarding all fat tissue and extraneous connective tissue and blood
vessels, to leave only a smooth serous side and a fibrous side. The
fibrous side is thinned down to a maximum of 0.5 mm using pathology
scalpels. The dissected tissue is cut into smaller pieces of usable areas.
This tissue, which retains its natural structure, i.e. is not comminuted
or disintegrated and reconstituted, is submerged in a suitable container
of 0.2% phosphate buffered glutaraldehyde pH 7.4 and kept at room
temperature. The submerged tissue is laid flat in the container and left
unstressed. The container is kept closed to eliminate the possibility of
contamination to the tissues, and Good Laboratory Practice Regulations and
Good Manufacturing Practice Regulations are followed at all phases of the
process. After 24 hours has elapsed, the tissue is turned and the solution
discarded and fresh 0.2% buffered glutaraldehyde is added until the tissue
is completely submerged. This procedure is repeated at 48 and 72 hours.
After 72 to 96 hours, samples of the crosslinked tissue are tested using
standard Shrinkage Temperature testing apparatus and procedures to assure
adequate crosslinking. The crosslinked tissue is aseptically dissected to
final configuration under sterile environment, such as, for example, a
Class 100 Laminar Flow Bench. The final dimensions will depend upon the
particular patient and procedure for which the tissue is being prepared. A
series of tissues range in size may be prepared thus permitting the
surgeon to select the appropriate size. The surgeon can, of course, modify
a given size to meet a particular requirement as determined during
surgery. The tissue is inspected by Quality Assurance to assure compliance
with all specifications, packaged in an approved container of sterile
physiologic saline and radiation sterilized and treated as described.
There are, of course, many variables which are controlled according to well
known principles and prior art practices, and which may be adjusted and
varied without departing from the scope of this invention.
INDUSTRIAL APPLICATION
The tissues of the invention are suitable for shipment and sale as human
implants.
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