Treatment of collagenous tissue with glutaraldehyde and aminodiphosphonate calcification inhibitor

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 comprising treatment with at least one surfactant prior to fixation, treatment with agents which inhibit calcification and agents which resist attack by phagocytic cells and optional treatment with stabilizing agents.

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.

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 
are normally kept in 4% formaldehyde solution until required for 
implantation. 
It is important to note that, before implantation, the valve may be rinsed 
with sodium borohydride solution or glycine (10 mg/ml) to remove 
formaldehyde. The removal of formaldehyde reduces tissue necrosis and 
enhances wound healing. 
A number of tissue valves were made from tissue treated in accordance with 
the procedure illustrated in the above Example and these valves were 
implanted in calves. The calves were slaughtered after two months and the 
valves examined. There were no signs of calcification and endothelial cell 
coverage was substantial as indicated in FIG. 6 of the accompanying 
drawings. These results demonstrate the substantial improvement achieved 
by the process of the present invention. 
The improved effects achieved by the process of the present invention are 
demonstrated by the results illustrated graphically in FIGS. 1 to 5 of the 
drawings and shown photographically in FIG. 6 of the drawings. 
Referring to FIG. 1 of the drawings, it will be seen that uptake of drug 
increases steadily for about 30 minutes at room temperature (25.degree. 
C.) reaching a maximum of about 2.9% without a surfactant pretreatment. 
Further soaking time and increase of temperature, up to 37.degree. C., has 
very little effect on the drug uptake. 
Treatment of the tissue with surfactant according to the process of the 
invention, indicated by curves (S), not only increases the uptake time, up 
to 60 minutes, but also increases the amount of drug taken up by the 
tissue. Furthermore, the drug uptake is more temperature dependent and 
treatment at 37.degree. C. increases the drug uptake by 2% over that 
achieved at 25.degree. C. 
FIG. 2 illustrates the effect of fixation time and temperature on drug 
uptake. It is to be noted that increased fixation time does not 
necessarily increase the drug uptake. A fixation time of 24 hours appears 
to be optimum for maximum drug uptake up to a drug treatment time of two 
hours. However, increasing the temperature of fixation, from 25.degree. C. 
to 37.degree. C., provides a significant increase in drug uptake. 
FIG. 3 illustrates the effect of multiple cross-linking, i.e. repeated 
fixation treatments, on drug uptake. Starting at time 0 with a tissue 
initially fixed in glutaraldehyde according to the procedure described 
hereinabove, this graph illustrates the effect of repeated drug 
treatments, D, of periods of 120 minutes each intersperced with repeated 
treatments with glutaraldehyde, G, represented by the arbitrary time 
periods ab, cd and ef. These fixation periods may vary from two or three 
up to about twelve hours, indicated by broken lines in the drawing, and 
naturally there is no drug uptake during these fixation periods. However, 
by following this repetitive procedure the effect is cumulative and a 
considerable increase in drug intake is achieved. It will be noted that 
the curve for the drug uptake at the end of the first treatment period has 
almost flattened out and the subsequent increases achieved by following 
the stated repetitive procedure is totally unexpected and certainly not 
attained or attainable by the procedures disclosed in the prior art. 
FIG. 4 illustrates the effect of pH on drug (DP) bonding followed by 
reduction with sodium borohydride. It will be seen that bonding trends to 
increase with more alkaline solutions. 
The effect of the initial surfactant treatment in enhancing the binding of 
the drug to pericardial tissue is illustrated in FIG. 5. The percentage of 
drug conjugated to the tissue was determined by use of radioactive tracers 
and the tests were conducted to compare the drug uptake achieved using (1) 
fresh pericardial tissue with no surfactant or fixation as a control; (2) 
pericardial tissue fixed with glutaraldehyde (G.A.) after treatment with 
Triton X-100 (Tx); (3) pericardial tissue fixed with glutaraldehyde after 
treatment with polyoxyethylene (20) sorbitan monooleate, commercially 
available under the Trade Mark Tween (Tw); and (4) paricardial tissue 
fixed with glutaraldehyde after treatment with sodium dodecyl sulfate 
(SDS). 
It will be seen that an significant increase in drug uptake is achieved 
following the pre-treatment with sodium dodecyl sulfate, an anionic 
surfactant. This effect is even further enhanced by using a mixture of 
anionic and non-ionic surfactants according to the preferred embodiment of 
the invention. 
FIG. 6 shows the substantially complete coverage of the tissue surface with 
endothelial cells achieved after an implantation time of only two months. 
This effect serves to demonstrate the success of the implant and, in 
particular, the achievement of the advantageous results discussed herein 
with regard to the substantial absence of thrombosis, calcification, 
infection or degeneration to a degree not achieved in the prior art.