Sterilization of tissue implants using iodine

The present invention provides an iodine-based solution, and a method of using that solution, which sterilizes tissue implants without denaturing the proteins in the implant and without inducing calcification of the implant in vivo. Preferably, the tissue implants sterilized using the present invention are fixed without using glutaraldehyde. Most preferably, the tissue implants are fixed by photooxidation.

BACKGROUND OF THE INVENTION 
Many types of implantable devices for repairing or improving the function 
of human body parts are known. Examples are vascular prostheses and 
grafts, tissue valves, and even completely artificial organs. The material 
that is used to make these implants or prostheses may be synthetic or it 
may be actual tissue derived from man or from some other species. For 
example, tissue implants often are derived from porcine or bovine sources. 
When an implant is made of actual tissue, the tissue may be used fresh 
from the donor; however, it is preferable to preserve the implant tissue 
for later use. 
One primary obstacle to successful implantation of actual tissue implants 
is immune response against the implant by the recipient. Immune response 
against an implant is a result of antigenic differences between cells of 
the recipient and cells of the implant material. The recipient's natural 
immune response is to attack the foreign antigens on the cells of the 
tissue implant. 
One widespread means used to overcome immune reactions against a tissue 
implant is to fix and preserve the tissue implant using glutaraldehyde 
before implantation. Theoretically, glutaraldehyde is believed to coat, 
bind and cross-link the antigens on the surface of the tissue implant. As 
a result, the number of antigens on the implant that are capable of 
inducing an immune response in the recipient are reduced. 
Glutaraldehyde-preserved tissue implants are relatively inert biologically 
and have demonstrated long-term durability in some instances even though 
the glutaraldehyde renders them somewhat cytotoxic. However, 
glutaraldehyde treated implants also have demonstrated serious drawbacks, 
such as tissue-fatigue and a propensity toward calcification. 
Glutaraldehyde tends to leach out of a tissue implant into both the 
surrounding tissue and into the bloodstream. Also, because glutaraldehyde 
is cytotoxic, the cells exposed to the leached glutaraldehyde can be 
damaged. Cells damaged by glutaraldehyde often die and/or rupture. Dead 
and/or ruptured cells often serve as a nidus for calcification. In fact, 
calcification has proven to be one of the primary reasons for failure of 
glutaraldehyde-treated implants. 
One solution to this calcification problem has been to fix and preserve 
tissue implants using photooxidation rather than glutaraldehyde. 
Photooxidation involves placing the tissue implant in saline, exposing the 
implant to a photocatalytic dye, and then subjecting the implant to 
fluorescent light. Photooxidation also modifies the structure of the 
collagen and appears to provide new cross-links in the collagenous tissue. 
However, implants that have been fixed using photooxidation do not exhibit 
the same tendency to calcify as glutaraldehyde-treated implants. 
Although photooxidative fixing of tissue implants shows great promise, the 
implant still must be sterilized before it can be implanted in the 
recipient. Unfortunately, the most common method used to sterilize a 
tissue implant is to treat the implant with glutaraldehyde. Sterilization 
with glutaraldehyde, even after the tissue implant has been fixed, still 
could create a calcification problem. Therefore, it would be advantageous 
if tissue implants could be sterilized without using glutaraldehyde. 
Historically, many germicidal or disinfectant solutions have been used to 
sterilize various objects and materials. The majority of such solutions 
have been used to disinfect solid surfaces. However, some disinfectant 
solutions have been used to disinfect soft surfaces, including human skin. 
Some of the disinfectant solutions previously used to sterilize human 
tissue, for example, the preparation known by the trademark 
"Betadine.TM.," have been iodine-based. Although iodine-based 
disinfectants have been used safely and effectively to sterilize the 
surface of living tissue, iodine-based solutions have not been used to 
sterilize non-living tissue such as the tissue found in a tissue implant. 
Whether or not an iodine-based disinfectant solution can safely and 
effectively sterilize the non-living tissue in a tissue implant is a valid 
concern. Living tissue can survive relatively rigorous conditions because 
living tissue is capable of repairing any damage that may result from such 
conditions. In contrast, non-living tissue cannot repair itself. When the 
proteins in non-living tissue are subjected to rigorous conditions, they 
tend to denature. Denaturation of the protein in the tissue implant, which 
cannot be repaired by the non-living tissue, detrimentally affects the 
physical properties of the tissue implant. 
A method of sterilizing tissue implants which does not cause protein 
denaturation and which does not induce calcification in vivo would be 
highly desirable. 
SUMMARY OF THE INVENTION 
The present invention provides an iodine-based solution, and a method of 
using that solution, which sterilizes tissue implants without denaturing 
the proteins in the implant and without inducing calcification of the 
implant in vivo. Preferably, the tissue implants sterilized using the 
present invention are fixed without using glutaraldehyde. Most preferably, 
the tissue implants are fixed using photooxidation.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention involves the sterilization of tissue implants which 
preferably have not been treated with glutaraldehyde. Most preferably, the 
tissue implants used in the present invention will have been fixed 
photooxidatively using the procedure described in U.S. patent application 
Ser. No. 07/388,003. 
In a preferred embodiment of the present invention, the tissue implants are 
sterilized using an iodine based germicidal solution under conditions 
having four primary variables: time of incubation; temperature of 
incubation; chemical content; and, pH of the germicidal solution. If any 
one of these four parameters is too stringent, the tissue implant may be 
damaged or the sterilization may be insufficient. Therefore, these 
parameters must be carefully controlled. 
Although it is possible not to add stabilizing salts to the iodine based 
solution of the present invention, the solution preferably contains iodine 
stabilizing salts which are believed to help maintain the elemental iodine 
in a microbiocidal condition. A number of salts might be suitable for this 
function, preferably halide salts, and most preferably iodide salts, such 
as potassium and sodium iodide. 
Another variable which tends to increase the microbial kill efficacy of the 
present solutions is to refresh the solution at approximately 24 hour 
intervals. If the solution is refreshed, solutions containing lower 
concentrations of iodine appear to be more efficacious. 
In order to avoid damaging the tissue implants, as the stringency of any 
one of the following treatment parameters is increased within the 
following ranges, the stringency of the other parameters should be 
decreased: 
______________________________________ 
Parameter Low High 
______________________________________ 
elemental iodine 0.01% 0.20% 
(I2) 
potassium iodide 0% 0.40% 
(KI) 
sodium iodide (NaI) 
0% 0.40% 
ethanol 0% 20% 
pH (aqueous 5.0 6.8 
solution) 
NaCl -- 4M 
time of incubation 
3 hours 2 weeks 
incubation 20.degree. C. 
50.degree. C. 
temperature 
______________________________________ 
A preferred embodiment of the present invention involves incubation of a 
tissue implant at a temperature between about 35.degree.-39.degree. C. for 
a period of time sufficient to sterilize the implant, typically between 
about 46-50 hours, in a solution containing between about 0.09-0.11% each 
of elemental iodine, potassium iodide, and sodium iodide and between about 
1.8-2.0% ethanol, the solution having been buffered to a pH between about 
6.4-6.6 using purified water and phosphate buffered saline. The ethanol 
content of the solution described in these preferred embodiments is a 
result of residual ethanol from the solution used to prepare the iodine 
stock. Although the present invention is functional if additional ethanol 
is present, no additional ethanol is added in these preferred embodiments. 
A particularly preferred embodiment of the present invention involves 
incubation of the implant at approximately 37.degree. C. for about two 
days in a solution containing about 0.1% each of elemental iodine, 
potassium iodide, and sodium iodide, about 1.9% ethanol, the solution 
having been buffered to a pH of about 6.5 using purified water and 
phosphate buffered saline. 
In a preferred method of preparing the germicidal solution of the present 
invention, a concentrated (10X) phosphate buffered saline solution ("PBS") 
is prepared by combining about 76 gm NaCl, 35 gm Na.sub.2 HPO.sub.4, and 
approximately 950 mL distilled or purified water (to a total volume of one 
liter), and stirring the resulting mixture until the ingredients are 
dissolved. Additional NaH.sub.2 PO.sub.4 is added (approximately 13.6-14 
gm) until the pH of the resulting solution is about 6.5. Unless otherwise 
specified, the chemicals used herein may be obtained from a number of 
commercial sources, such as Sigma Chemical Company, St. Louis, Mo., or 
Aldrich, 1001 West Saint Paul Avenue, Milwaukee, Wiss. 53233. 
A preferred iodine stock solution is prepared by combining about 5.0 gm 
each of elemental iodine (I.sub.2), potassium iodide, and sodium iodide, 
with about 100 ml of 95% ethanol which has been prewarmed to about 
37.degree. C. The mixture is swirled until the iodine is completely 
dissolved. The resulting solution may be stored in the dark for up to one 
month. 
To prepare the solution for actual use to sterilize a tissue implant, the 
following should be dissolved in about 850 mL distilled or purified water 
in a 2 liter beaker or flask: about 100 mL 10X PBS stock solution; 20.0 mL 
5% iodine stock solution. After all of the components have dissolved, the 
pH of the solution should be adjusted to about 6.5 using the appropriate 
sodium mono- and dibasic-phosphates, as needed. The solution then should 
be brought to 1.0 liter using additional distilled or purified water, as 
needed. The resulting solution should be filtered through a 0.2 micron 
sterilizing filter into a sterile container which may be capped, sealed, 
labeled, and stored at room temperature in the dark for up to a maximum of 
one week. 
In order to sterilize a tissue implant using the foregoing iodine solution, 
the following preferred procedure should be performed within a laminar 
flow hood. All personnel should wear appropriate attire, including gowns, 
mask, hat, and sterile gloves. The following items should be placed within 
the laminar flow hood: sterile 5 inch forceps; clean plastic jars; 
prepared labels; prepared iodine solution. The implant container should be 
opened and the tissue implant and test sample or "swatch leaflet" removed 
using the sterile forceps. The containers should be opened one at a time 
and the transfer should be completed before another tissue implant is 
removed from another container. The tissue implant and swatch leaflet 
should be placed in a clean plastic jar (such as a 3 oz. polypropylene 
jar) and the prepared iodine sterilant should be poured into the jar until 
the solution completely covers the valve (approximately 100 mL). The Jars 
containing the implant, the test swatch, and the iodine solution should be 
closed tightly and the outside of the jar should be wiped with a lint free 
cloth and ethyl alcohol and labelled. Two empty jars then should be filled 
with approximately the same volume of iodine and labeled as controls, and 
all of the jars should be placed in an incubator which has been calibrated 
to the desired temperature, preferably about 37.degree. C., and incubated 
for 48.+-.2 hours, or for 24 hour increments after which the iodine 
solution was refreshed. 
After incubation, the jars should be removed from the incubator and placed 
in the laminar flow hood, along with a sterile five inch forceps, clean 
plastic jars, labels, and sterile 50% ethanol. The container should be 
opened and the tissue implant and swatch leaflet removed from the 
iodine/ethanol solution using the sterile forceps. The containers should 
be opened one at a time. The implant should be placed into one clean jar 
and the swatch leaflet placed into another, and 50% ethanol should be 
aseptically poured into the jars until the implant and the swatch leaflet 
are covered completely (approx. 100 mL). The jars then should be closed 
tightly, and the outside of the jar should be wiped with a lint free cloth 
and ethyl alcohol and labeled. Two control jars for later sterilization 
testing then should be prepared by filling two empty jars with 
approximately the same volume of 50% ethanol and labeling the jars 
"control." 
As an alternative to removing the tissue implant and swatch leaflet from 
the sterilization solution, it also may be possible to simply leave the 
implant and leaflet in the same solution for shipping, or to add ethanol 
to the sterilization solution without changing containers or removing the 
sterilization solution. 
Tissue implants that have been treated according to the foregoing preferred 
method should be sterile and ready for implantation. The foregoing 
preferred parameters were derived as a result of three basic types of 
tests. A first type of test measured the biophysical integrity of the 
tissue implant after sterilization according to the present invention. 
Another type of test measured the biochemical integrity of the tissue 
implant. A third type of test measured the effectiveness of the regimen in 
sterilizing the implant. A fourth type of test involved actual 
implantation and monitoring of the implants. 
A simple examination with the naked eye is capable of detecting some 
biophysical damage to tissue, e.g., damaged tissue Generally appears 
tough, leathery, or curled. Another, more precise method of measuring 
biophysical integrity is a "shrink" test. 
Shrink tests are based on the following phenomenon. When bovine or porcine 
tissue is placed in water and heated, healthy tissue typically begins to 
shrink or shrivel at a temperature of around 62.degree. C. If the tissue 
has lost integrity or been damaged in some way, e.g., by exposure to acid, 
the damaged tissue will begin to shrink at a lower temperature. The 
"shrink temperature" (T.sub.s), or the temperature at which the tissue 
begins shrinking, can be measured by extending the tissue between the two 
ends of an extensometer and measuring the temperature at which the tissue 
begins to shrink. 
In addition to shrinking at lower temperatures, damaged tissue may exhibit 
several other symptoms: the thickness of the tissue may change; the tissue 
may become more susceptible to attack by destructive agents, such as 
proteases, other digestive enzymes or chemical agents; the tissue may 
exhibit leakage of tissue components, such as collagen; and, well-defined 
striation of the tissue may begin to disappear when the tissue is scanned 
with a transmission electron microscope. Thus, tests were conducted which: 
(1) measured the percent change in thickness of the tissue before and 
after treatment according to the present invention; (2) subjected the 
treated tissue to a solution containing the chemical digestive agent 
cyanogen bromide (CNBr), which is capable of digesting damaged tissue; (3) 
subjected the treated tissue to an assay for collagen extractability 
(PAGE); and, (4) subjected the treated tissue to scanning by transmission 
electron microscopy (TEM). 
Tests also were conducted to measure how effective various treatment 
regimens were in actually sterilizing tissue implants. A tissue implant 
may be contaminated in three separate locations: the components of the 
implant; the germicidal solution, itself; and, the ethanol in which the 
implant is stored after it is sterilized. Each of these locations was 
tested for contamination after tissue implants had been sterilized using 
the present invention. 
Finally, tissue implants which had been sterilized according to the present 
invention actually were implanted in juvenile sheep and later explanted 
and examined for integrity and evidence of calcification. 
In most of the following tests, the sterilization solution was prepared by 
a slightly different procedure than the preferred procedure already 
described; however, the procedure resulted in the same sterilization 
solution. The variation in procedure involved adding the potassium and 
sodium iodides after the iodine stock solution had been formed. The iodine 
stock solution was prepared by combining the elemental iodine (I.sub.2) 
with 95% ethanol which was prewarmed to about 37.degree. C. The mixture 
was swirled until the iodine is completely dissolved. The sterilization 
solution then was prepared by dissolving in distilled purified water: the 
10x PBS stock solution; the 5% iodine stock solution prepared as just 
described; sodium iodide; and, potassium iodide. 
The following tests demonstrated that the present invention safely and 
effectively sterilized tissue implants without inducing the calcification 
normally seen in Glutaraldehyde-treated implants. 
INTEGRITY TESTS 
SHRINK TESTS 
Incubation of bovine pericardial tissue in a composite iodine solution can 
result in altered shrink temperature characteristics. The following 
experiments indicated that only very mild conditions result in an 
unchanged shrink temperature value (typically 65.degree..+-.2.degree. C.). 
These conditions (e.g . 1 day in 0.1% iodine at room temperature) may be 
insufficient to provide an adequate sterilization process for a tissue 
valve. However, more extensive iodine treatment conditions (e.g. longer 
times, higher concentrations, higher temperatures) can lead to markedly 
lower shrink temperature values. Tissue exhibiting a T.sub.s in the 
55.degree. C. or lower range are visually altered, appearing thicker, 
curled, and discolored. Between these extremes exists a broad window of 
tissue treatment conditions. Many tissue treatment conditions presented 
here result in a lowering of shrink temperature values of only 
2.degree.-3.degree. C., which may not indicate tissue damage. Beyond this 
plateau, lie more extreme conditions resulting in T.sub.s values from 
55.degree.-62.degree. C. Note that even control solution treated tissue 
(no iodine) exhibits a 2.degree.-3.degree. C. drop in Ts values. 
Therefore, a similar drop in T.sub.S under iodine treatment conditions 
indicates the absence of any iodine mediated tissue damage. 
Safe tissue treatment is arbitrarily defined here as resulting in tissue 
with a shrink temperature of 62.degree. C. or greater. From the following 
experiments, one limit, or window, of safe tissue treatment is defined as 
a set of conditions (with a reasonable safety factor) with maximal 
parameter values of about: 
0.1% iodine 
37.degree..+-.2.degree. C. incubation temperature 
4 days incubation, and 
pH 6.0 (minimum) 
with the following acceptable additions of: 
iodide salts 
NaCl, or 
ethanol 
The use of iodine as an effective sterilant depends on a suitable quality 
of tissue emerging after sterilization. If tissue presenting T.sub.s 
values above 62.degree. C. is deemed acceptable, there exists a range of 
incubation conditions demonstrated here which have the properties of: 
consistently resulting in this quality of tissue 
a reasonable safety margin for T.sub.S values, and 
an expectation of acceptable sterilization capabilities 
Experimental Procedure 
In the following experiments, photooxidized bovine pericardial tissue was 
destained to a light blue to remove excess photocatalyst, as might occur 
in tissue valve product, and cut into one inch squares by water jet before 
use. The tissue pieces were soaked in a total of 40-80 mL of iodine (or 
control) solution per square in a clean polypropylene jar. Iodine 
solutions were prepared as described above, with the PBS solution diluted 
to 1X PBS and the pH adjusted with monobasic potassium phosphate after 
iodide/iodine addition. 
After incubation with iodine or control solutions, tissue samples were 
transferred to 50% ethanol/water and stored at 4.degree. C. until 
analysis. Solution color and pH were noted. Tissue squares were cut into 
one-half by one inch rectangles and briefly hydrated with water at room 
temperature before initiation of a shrink test similar to those known in 
the art. See, e.g., Nimni, M. "Collagen: Structure, Function, and 
Metabolism in Normal and Fibrotic Tissues." Seminars in Arthritis and 
Rheumatism, Vol. XIII, No. 1 (August 1983), incorporated herein by 
reference; Flandin, F., Buffevant, C., and Herbage, D. "A Differential 
Scanning Calorimetry Analysis of the Age-Related Changes in the Thermal 
Stability of Rat Skin Collagen." Biochimica et Biophysica Acta 791 (1984) 
205-211; and Le Louis, M. , et al. Connect. Tiss. Rsch. 9 (1982) 253-262, 
incorporated herein by reference. 
The following procedure was performed. A water bath was filled to within 2" 
of the top, and the heater was turned on. Extensometers were plugged into 
their respective positions at the back of the tester. Each extensometer 
was mated with an appropriate channel, and a thermocouple and a computer 
supplied with an appropriate program were attached. The shrink tester 
program was retrieved and program prompts were followed for calibration, 
sample identification, and sample placement. To calibrate the minimum 
shrink, extensometer #1 was placed between the two rightmost calibration 
pegs such that the extensometer was firmly pressing against the sides of 
the pegs and the support on the bottom. To calibrate the maximum shrink, 
extensometer #1 was placed between the two outermost calibration pegs such 
that the extensometer was firmly pressing against the sides of the pegs 
and the support on the bottom. The calibration was made and the 
calibration curve was compared to the previous calibration curve. The 
curves were consistent; therefore, the samples were placed on the 
extensometers by penetrating one end of the samples with one side of the 
extensometer and then penetrating the other end of the samples so that the 
samples were pulled taut. The maximum desired test temperature was 
entered, and the extension of the sample was measured for each specimen. 
Then, the samples were immersed in the water bath and heated to the test 
temperature at a programmed rate. 
The data reported here represents the inflection point of tissue shrinkage, 
i.e., the point where the tissue begins rapid shrink. Most data points 
represent duplicate measurements. 
The following solution compositions for sterilants were used, and samples 
were treated at 42.degree..+-.2.degree. C. for 2 days, unless otherwise 
noted. 
Solution B: 
10% ethanol 
0.1% iodine (each of NaI, KI, and I.sub.2) in water 
Solution C: 
0.1% iodine (each of NaI, KI, and I.sub.2) 
1X PBS 
Solution D: 
10% ethanol 
0.1% iodine 
1X PBS 
Note that the I.sub.2 stock (50X) is prepared with 95% ethanol. Therefore, 
a 0.1% iodine solution contains an additional 1.9% ethanol. Also, residual 
ethanol invariably is carried over from the tissue samples to be 
sterilized (they are stored in 50% ethanol). 
General Observations 
Stability of pH 
After transfer of the tissue samples to ethanol, the remaining iodine 
solutions were retained for color and pH analysis. It was found that the 
pH of buffered solutions lowered only slightly upon incubation, generally 
less than 0.1 pH units. The most dramatic shifts (0.3 pH units lower) were 
seen with samples incubated at an extreme of 42.degree. C. for 16 days. 
These relative pH stabilities were encouraging signs as low pH has been 
shown to be harmful to the tissue, and projected sterilization regimens 
involved extended incubations. 
Color of Iodine Solutions 
A typical 0.1% iodine solution has a deep red color. After incubation under 
a variety of conditions, the color varies from the same deep red to clear 
or even a pale blue or green if dye-stained tissue is used. In the 
experiments described here, a general trend with respect to solution color 
was noted. The color of the iodine solution was more faded under 
incubation conditions of higher temperature, higher pH, and longer 
incubation time. Under an "average" set of conditions (Solution C), namely 
0.1% iodine, pH 6.5, incubated at 42.degree. C. for two days, solutions 
were a pale red/deep yellow. Earlier results supported a hypothesis that 
the remaining "redness" of the sterilant after specific incubation times 
was proportional to the killing effectivity of the solution. This does not 
indicate, however, that conditions leading to solution fading are 
necessarily detrimental since the total antimicrobial action of the 
solution may be sufficient regardless of the eventual iodine dissipation. 
In other words, it may not matter if the solution is clear after the 
incubation cycle as the solution (and product) already may be sterile. 
Tissue Appearance 
Tissue appearance may be used as a gross predictor of shrink temperature. 
Note that the following observations refer to tissue sample appearance 
prior to the actual shrink temperature analysis of those samples (shrink 
temperature determination is a destructive analytical tool which leaves 
tissue curled, tough, and approximately one-fifth of its original size). 
Samples exhibiting low shrink temperatures (below 55.degree.-60.degree. 
C.) tend to appear thicker, curled, less flexible, and sometimes darker. 
Samples with moderately low shrink temperatures (60.degree.-62.degree. C.) 
are difficult to distinguish from non-sterilized photooxidized tissue 
except for iodine staining (which is reversible upon storage in 50% 
ethanol). In these experiments, conditions leading to low shrink 
temperature also resulted in more dramatic tissue changes. Conditions of 
higher temperatures, lower pH, higher iodine levels, and longer incubation 
times resulted in darker, more rubber-like tissue samples. 
Shrink Temperature Measurement 
Shrink test data were gathered as described. Control samples, either fresh 
or photooxidized, but not treated with any iodine solution, typically 
exhibited a shrink temperature inflection at 66.degree..+-.2.degree. C. 
These data are not shown; however, note control samples in various 
experiments which were incubated in solutions and treated under 
sterilization conditions in the absence of any iodine (shown with 
individual experiments as no iodine) or not incubated (shown as t=0 data). 
Percent Change of Thickness Measurement 
The percent change of thickness of the tissue was measured by first 
ensuring that, without a sample present (resting state), the thickness 
gauge (Federal Products, Providence, RI, Model 691B-R2 equipped with 50 g 
total load) reads 0.+-.0.0001 inch. The gauge lever then was depressed to 
raise the load face. With the lever depressed, the flattened, unwrinkled 
sample was placed under the load face and on top of the load platform. The 
lever was raised until the face fully rested on the sample. The load was 
allowed to settle on the tissue for a period of thirty seconds, and the 
reading was recorded. 
Experiment 1 
Parameters 
Incubation time=2 days 
No ethanol 
Incubation temperature=23.degree., 30.degree., 37.degree., 40.degree., 
43.degree., or 46.degree. C. 
pH=5.0, 5.8, 6.2, 6.5, 6.8, or 7.2 
iodine level=0.1% 
36 samples 
In this initial parameter study, one piece of tissue was incubated per 40 
mL solution for 2 days at one of six temperatures (listed above), and at 
one of six pH values (listed above). These solutions were made in the 
absence of any ethanol. As seen in FIG. 1, there is a general trend 
towards lower shrink temperatures with higher temperatures or lower pH 
values. The samples incubated at 43.degree. C. in the two lower pH 
solutions were severly curled and rubbery and shrink temperature analysis 
yielded no useful data. 
Experiment 2 
Parameters 
t=2 days 
.+-.ethanol 
temp.=37.degree. or 46.degree. C. 
pH=5.8 or 6.5 
NaI:KI:I.sub.2 =1:1:1, 2:2:1, or 4:4:1 
I.sub.2 =0.1% 
24 samples 
These samples were tested to determine the effect, if any, of additional 
iodide salts on tissue. It was hypothesized that additional iodide salts 
might help to establish an equilibrium in which elemental iodine remained 
in an antimicrobial form. Thus, lower levels of total elemental iodine 
might be used to effect the same antimicrobial activity in the presence of 
additional iodide salts. Commercial tincture of iodine solutions typically 
have an iodide/iodine ratio exceeding unity. Also, the extra salt (higher 
ionic strength) might have an additional protective effect on the tissue. 
However, as seen in FIG. 2, additional iodide salts exerted no apparent 
protective effect on tissue. Likewise, no negative effects were noted. If 
deemed necessary, the use of these increased ratios of iodide/iodine would 
not be expected to harm the tissue and in fact might provide a more 
effective iodine sterilant. 
The color of different iodine solutions indicated a retention of iodine 
color at higher iodide levels, although these solutions were only slightly 
more intense in color (in 5 of 8 "sets," the highest iodide-containing 
solutions were the most colored, less colored in one, and 
indistinguishable in the other two). This might indicate increased 
stability of elemental iodine in the presence of additional iodide salts. 
However, incubation temperature was much more of a determinant in the 
final color of the sterilant, with those solutions incubated at a higher 
temperature being less colored in all cases. 
Experiment 3 
Parameters 
t=2 days 
iodine=0, 0.01, 0.02, 0.05, or 0.1% (each) 
temp.=23.degree., 37.degree., 42.degree., or 46.degree. C. 
pH=5.0 or 6.5 
40 samples (includes 0% iodine controls 
This experiment focused on the treatment of tissue with lower amounts of 
iodine. The ratio of iodide salts to iodine remained at 1:1:1. To 
correspond with lower iodine levels, other parameters were adjusted to 
test the limits of tissue stability, namely higher temperature and lower 
pH, both of which should have lead to greater microbial activity. As seen 
in FIG. 3, there was a gradual decrease in shrink temperature values as 
the levels of iodine increased. These values remained stable at pH 6.5 at 
all iodine levels up to 37.degree. C., yet fell below 62.degree. at 
43.degree. C. or above (FIG. 3A). The same statements can be made 
concerning the pH 5.0 samples although there was a sharper drop in values 
(FIG. 3B). FIGS. 3C and D, in which the same data was plotted vs. 
incubation temperatures, reemphasize the drop in shrink temperature at 
elevated temperatures. Again the effect was seen to be more dramatic at pH 
5.0 (FIG. 3D). 
Experiment 4 
Parameters 
time=0, 1, 2, 4, 8, or 16 days 
temp.=22.degree., 37.degree., or 42.degree. C. 
pH=5.8 or 6.5 
iodine=0.1% 
ethanol=.+-.10% added 
72 samples+controls (no iodine) 
"Milder" parameters with respect to tissue shrink temperature effects 
(lower temperature and higher pH) might not lead to optimal antimicrobial 
activity. However, extending the time of iodine exposure might 
counterbalance this effect. This experiment was designed to test the 
effect of extended exposure to a variety of iodine solutions on tissue 
shrink temperature. FIGS. 4A and B indicate a decline in values over time 
under most conditions tested. The negative effects were less dramatic in 
the presence of ethanol, suggesting a protective effect. However, earlier 
experiments have indicated a lower kill effectivity in the presence of 
alcohol. Note, however, that the conditions under which this effect was 
most pronounced were those least likely to be employed, i.e., lower pH and 
higher temperature. In addition, these conditions lead to generally lower 
shrink temperatures overall. 
The results of the control solution (in FIG. 4A, no iodine) containing no 
iodine at pH 5.8 leads to two conclusions. First, the presence of iodine 
itself can cause a lower shrink temperature value (vs. the control) and, 
secondly, there is a 2-3 degree drop in T.sub.s which apparently is caused 
by the buffer and incubation conditions alone, and not by the iodine. This 
drop in T.sub.s, which is unrelated to iodine, may indicate that a 2-3 
degree change does not represent any tissue damage (it certainly does not 
reflect iodine-induced tissue damage) and, furthermore, may be 
inconsequential in regards to tissue performance. Therefore, under 
carefully controlled conditions where, in the presence of iodine, there is 
a drop of 2-3 degrees there also may be no reason to suspect any tissue 
damage. 
Experiment 5 
Parameters 
[NaCl]=2.5, 3.0, 3.5, or 4.0M 
pH=5.0, 5.5, 6.0, 6.5 
temp.=23.degree. or 40.degree. C. 
iodine=0.1% 
32 samples+controls 
Collagen stability is known to increase under high salt conditions. This 
experiment was designed to test whether high ionic strength solutions had 
any protective effect on the tissue. Similar shrink temperature values 
were obtained regardless of salt levels (from 2.5-4.0M). Overall, the 
values were lower with tissue incubated at 40.degree. C. and little effect 
was seen with pH differences. In summary, high salt did not reverse or 
prevent the typical 2-3 degree drop in shrink temperature that was 
predictable for 0.1% iodine incubation for 2 days. Neither did high salt 
negatively impact shrink temperature values. Since no negative control (no 
salt) was included, it was difficult to directly assess whether the salt 
had a protective effect at a lower pH (5.0 & 5.5). However, comparison 
with results from previous experiments at lower pH values indicates that 
the salt may have had a slight protective effect. 
Experiment 6 
TEM Analysis 
Transmission electron micrographs of tissue treated according to the 
present invention were obtained using known procedures, and the results 
are shown in FIG. 5. FIG. 5 graphically illustrates the potential harm of 
an extreme iodine sterilant. FIG. 5A shows a tissue treated in Solution B 
(pH .about.3-5, 0.1% iodine, 2 days, 42.degree. C.). Note the unravelling 
of collagen fibrils indicating severe base tissue damage. Contrast this to 
tissue in FIG. 5B, representing tissue treated using the following 
parameters, in which the collagen fibrils appear intact: 
Parameters 
0.1% iodine 
pH 6.5 (PBS buffered) 
"no" ethanol (except 2% from iodine stock and residual from tissue) 
2 days incubation at 42.degree. C. 
Despite these results, this condition may not be desired due to its 
proximity to the "shrink temperature ledge". 
Experiment 7 
Animal Implants 
Prior to discovering apparent tissue damage by Solution B (pH .sup..about. 
3.5, 0.1% iodine, 2 days, 42.degree. C.), several valves were sterilized 
by this method and implanted in sheep. Accompanying tissue swatches 
revealed shrink temperature data for this tissue of 58.degree.-60.degree. 
C. for three of these valves and a value of 65.degree. C. for the other. 
One of these animals (with an accompanying swatch T.sub.s of 58.3) died of 
unknown cause at day 66. Three animals were sacrificed and all of the 
valves looked to be in excellent condition with slight tissue stretching 
at 5 months. Angiography of each animal prior to sacrifice indicated 
physiologic performance for all valves with no regurgitation. The 
accompanying tissue swatches for these animals exhibited shrink 
temperatures of 59.6.degree., 59.4.degree., and 65.8.degree. C., 
respectively. Thus, despite some signs of tissue damage, Solution B 
treated valves appeared to perform adequately in vivo. 
DIGESTION AND LEAK TESTS 
Experiment 8 
In the following experiment, a 0.1% iodine solution was effective as a 
sterilant against 1.times.10.sup.7 B. subtilis spores in the presence or 
absence of tissue valve components, while a 0.01% iodine solution was 
effective only in the absence of components. The effect on the tissue 
appeared to be minimal, with only a slight decrease in shrink temperature 
values and little effect on cyanogen bromide digestion, protein 
extractability, or tissue thickness. Most components were stained by the 
iodine solutions, although this appears to be reversible upon storage in 
50% ethanol. 
Two separate iodine sterilization solutions were used in the following 
experiments: 
Condition 1: 
0.1% iodine 
phosphate buffered to pH 6.5 
incubation at 37.degree..+-.2.degree. C. 
incubation for 48.+-.2 hours 
Condition 2: 
0.01% iodine 
phosphate buffered to pH 6.0 
incubation at 42.+-.2 hours 
incubation for 48.+-.2 hours 
After incubation, samples were aseptically transferred to sterile filtered 
50% ethanol. 
The materials used in the incubations were dictated by the expected 
quantities and types of materials found in a completed tissue valve, which 
include: photooxidized bovine pericardial tissue; photooxidized porcine 
pericardial tissue; silastic; Reemay cloth; Dacron cloth; Elgiloy; 
polypropylene jars and lids; iodine solution; sutures (Gore-Tex and 
Green); valve holders; and, Teflon tags. The following types of tests were 
performed: 
material appearance of valve components (including jar and solution), 
cyanogen bromide (CNBr) digestion of tissue, 
collagen extractability of tissue (bovine and porcine), 
thickness measurement of bovine tissue, 
shrink temperature analysis of tissue (both), 
sterility of the iodine sterilants, 
sterility of the tissue samples, 
sterility of the ethanol storage solution, and 
sterility of the control solutions. 
Table 1 reflects the conditions of each sample. A "+" under the column 
entitled "Inoculated" indicates prior spore inoculation with 10.sup.7 B. 
subtilis. A "+" under the column entitled "Components" indicates the 
presence of tissue valve components. A "-" under either column indicates, 
respectively, the absence of prior spore inoculation or tissue valve 
components: 
TABLE 1 
______________________________________ 
Condition or 
Sample Solution Inoculated 
Components 
______________________________________ 
10 Condition 1 - + 
control (no 
iodine or iodide 
salts, pH 6.5) 
11 Condition 1 - + 
(0.1% iodine) 
12 Condition 1 - + 
(0.1% iodine) 
13 Condition 1 - + 
(0.1% iodine) 
14 Condition 1 - + 
(0.1% iodine) 
15 Condition 1 - + 
(0.1% iodine) 
16 Condition 1 + + 
(0.1% iodine) 
17 Condition 1 + + 
(0.1% iodine) 
18 Condition 1 + + 
(0.1% iodine) 
19 50% ethanol - + 
(RT inc.) 
IC+ Condition 1 + - 
(0.1% iodine) 
IC- Condition 1 - - 
(0.1% iodine) 
20 Condition 2 - + 
control (no 
iodine or iodide 
salts, pH 6.0) 
21 Condition 2 - + 
(0.01% iodine) 
22 Condition 2 - + 
(0.01% iodine) 
23 Condition 2 - + 
(0.01% iodine) 
24 Condition 2 - + 
(0.01% iodine) 
25 Condition 2 - + 
(0.01% iodine) 
26 Condition 2 + + 
(0.01% iodine) 
27 Condition 2 + + 
(0.01% iodine) 
28 Condition 2 + + 
(0.01% iodine) 
29 50% ethanol - + 
(42.degree. C. inc.) 
2C+ Condition 2 + - 
(0.01% iodine) 
2C- Condition 2 - - 
(0.01% iodine) 
______________________________________ 
Cyanogen bromide digestion: 
Cyanogen bromide digestion was conducted as follows: 
Reduction with 2-mercaptoethanol 
The amount of ammonium carbonate (Sigma A 8045) required for a 1M solution 
was calculated as follows: (78.06 gm/mol).times.(1 mol/1).times.final 
volume=gm of ammonium carbonate The calculated amount was added to 80% of 
the final volume of water and mixed on a stir plate until dissolved. The 
volume was measured in a graduated cylinder, and water was added to bring 
to the final volume and the resulting solution was mixed by inversion. The 
solution may be stored at 4.degree. C. in a tightly capped container. 
Tissue samples were cut to the desired size (typically 4.times.6 mm) using 
a scalpel, and weighed on a Mettler analytical balance. The weight values 
were recorded (typically 20-30 mg.), and each piece was placed in 
approximately 1 ml of Ozarka distilled water in a microcentrifuge tube for 
rinsing. 
The previously incubated tissue samples were transferred to clean 
microcentrifuge tubes, and to each tissue sample tube was added: 0.65 ml 
water, 0.1 ml 1M ammonium carbonate, and 0.25 ml 2-mercaptoethanol, to a 
final volume of 1 ml and a final concentration of 0.1M ammonium carbonate 
and 25% 2-mercaptoethanol (BioRad 161-0710). A desired amount (1 ml or 
less) of soluble collagen also was dispensed in microcentrifuge tubes. The 
same amount of ammonium carbonate and 2-mercaptoethanol was added to the 
soluble collagen, the final volume then was brought to 1.5 ml with water 
to result in the same final concentrations. All of the tubes then were 
incubated in a water bath at 55.degree. C. overnight. 
Removal of 2-mercaptoethanol 
The 2-mercaptoethanol was removed from the samples as follows: 
Tissue Samples 
Each tissue sample was rinsed three times in .sup..about. 5 ml 60% ethanol, 
or until the 2-mercaptoethanol odor was gone. 
Preparation of cyanogen bromide 
Cyanogen bromide stock solution was prepared by placing a bottle of 
cyanogen bromide (Sigma C 6388) in the fume hood. (Note: CNBr is extremely 
toxic and should be kept in the hood at all times. Protective clothing and 
eyewear should be worn when handling CNBr.) 70% formic acid was prepared 
using the following formula: 
(a) (0.70)(final volume).div.0.99=volume of 99% formic acid 
(b) (final volume)-volume of 99% formic acid=water 
(c) combine 99% formic acid and water. 
The 70% formic acid was added to the bottle for a concentration of 1 gm/ml 
(e.g., add 1 ml 70% formic acid to 1 gm CNBr). A tiny stir bar was placed 
in the bottle and the solution was stirred on a stir plate in the hood 
until the solid had dissolved. 
The tissue pieces were transferred to 7 ml white-capped plastic tubes. A 
desired amount of the soluble collagen samples (0.43 ml or less) was 
transferred to a clean microcentrifuge tube. 
The required amount of cyanogen bromide working solution was calculated by 
adding the weight (mg) of all samples for CNBr digestion (A) and 
multiplying the total value by five (5). The final concentration of each 
sample in solution should be 10 mg sample/ml. The required volume of 
working solution was calculated by dividing A/10 mg/ml=X ml. 
The CNBr working solution (50 mg/ml CNBr in 70% formic acid) was prepared, 
with 10% extra solution to insure an adequate amount, by determining X, Y, 
and Z, as described below, and multiplying by 1.1: 
The required amount (ml) of 99% formic acid to make X ml of 70% formic acid 
was calculated using the following equation: 
EQU (0.70)(X ml).div.(0.99)=Y ml 
The required amount of 1 gm/ml CNBr stock solution to make X ml of 50 mg/ml 
CNBr was calculated using the following equation: 
EQU (0.050 gm/ml)(X ml).div.1 gm/ml=Z ml 
Y and Z were combined in a graduated 50 ml FALCON tube, and water was added 
to a total of X ml. 
Digestion with CNBr 
The working CNBr solution was dispensed into the sample tubes while 
maintaining a concentration of 10 mg sample/ml working solution (e.g., 3 
ml CNBr working solution was added to a tissue piece weighing 30 mg using 
an Oxford 5 ml pipette). Each tube was capped tightly, placed in a test 
tube rack in the water bath, and incubated four hours at 30.degree. C. The 
water bath was turned off and the tubes were incubated at room temperature 
overnight. 
Similarly, 99% formic acid was added to the soluble collagen samples for a 
final concentration of 70% formic acid. E.g., using the following equation 
to calculate the amount of 99% formic acid to add for a final volume of 
1.5 ml: 
EQU (0.70)(1.5 ml).div.0.99=1.06 ml 
An appropriate amount of CNBr stock solution was added for a CNBr:(soluble 
collagen) weight excess of 5:1. Water was added to reach the desired final 
volume and to maintain a constant soluble collagen concentration for each 
of the samples (e.g., 0.5 mg/ml). Each microcentrifuge tube was closed 
tightly and placed in a floating tube rack in the water bath. The tubes 
then were incubated for four hours at 30.degree. C. The water bath was 
turned off and the tubes were incubated at room temperature overnight. 
Dilution of Samples 
Two microcentrifuge tubes containing 1.2 ml water each were prepared for 
each of the tubes containing a tissue sample. After overnight incubation, 
200 ul of each sample tube was added to each of the 2 microcentrifuge 
tubes and the contents were mixed by inversion. A 400 ul liquid sample was 
drawn and the appearance of the tissue noted. The sample tubes were stored 
at -20.degree. C. 
Microcentrifuge tubes containing enough water for a 5- to 10-fold dilution 
were prepared for each tube containing soluble collagen. After overnight 
incubation, an aliquot of each sample containing enough protein to 
visualize in one lane of a polyacrylamide gel was added to each of the 
microcentrifuge tubes, and the resulting contents were mixed by inversion. 
The sample tubes then were stored at -20.degree. C. 
Freeze Drying of Diluted Samples 
The Savant Speed-Vac SVC100 was placed in a fume hood and connected to a 
large (1 liter) Ehrlenmeyer flask using a vacuum tube and glass tubing 
inserted into a rubber stopper the size of the mouth of the flask. Another 
glass tube inserted into the same rubber stopper connected the flask to 
the vacuum pump with vacuum tubing. The flask was placed inside a 
styrofoam box, and the box was filled with dry ice. 2-propanol (99+%, ACS 
reagent grade) was added to create a cold slush around the flask. This is 
the cold trap, designed to freeze aqueous solutions under vacuum to avoid 
water accumulation in the vacuum pump oil. The cold trap was placed in the 
hood. 
All microcentrifuge tubes were frozen in the cold trap. With their caps 
open, all tubes were placed in the rotor of the Speed-Vac and maintained 
in proper balance. The Speed-Vac was started. The vacuum pump was turned 
on, and a strong vacuum pull was ensured by checking the seal on the lid 
of the Speed-Vac. The samples were dried for several hours, or until all 
liquid was gone and only residue remained. The vacuum was released before 
stopping the Speed-Vac. .sup..about. 1 ml water was added to each 
microcentrifuge tube, and each tube was vortexed and frozen again in the 
cold trap using the procedure just described. The residue of each set of 
tubes was combined as follows: 
If more than one microcentrifuge tube existed per sample tube, 500 ul of 
water was added to the first microcentrifuge tube of the set and vortexed 
well. Everything in the first tube was transferred to the subsequent tube 
of each set, vortexing well between each transfer. If only one 
microcentrifuge tube per sample existed, 500 ul water was added and the 
contents vortexed. The samples then were freeze dried once again in the 
cold trap, as just described. 
Sample Analysis 
Dry samples were dissolved in water or buffer for analysis. If stored 
before analysis, the samples were stored at -20.degree. C. The samples 
were analyzed by polyacrylamide gel electrophoresis. 
Collagen Extractability 
0.5M Tris-HCl, pH 6.8 and 10% SDS were prepared according to the BioRad 
PROTEAN II xi Slab Cell Instruction Manual (1990), incorporated herein by 
reference. 0.5% Bromophenol blue was prepared by dissolving 50 mg 
bromophenol blue in 10 ml of water purified using a MilliQ-UF filter 
system from Millipore Corp., Bedford, Mass. 2.5M NaCl was prepared by 
dissolving 7.3 gm NaCl in 50 ml MilliQ-UF water. 
2X Sample Buffer (2X SB) was prepared by combining the following to form 10 
ml total volume: 2 ml 0.5M Tris-HCl, pH 6.8; 2 ml 10% SDS; 2 ml glycerol; 
0.4 ml 0.5% Bromophenol blue; and 3.6 ml MilliQ-UF water. 
2 ml of extraction buffer was prepared as shown in the following chart: 
______________________________________ 
Sample Final Conc. Vol. (ml) 
______________________________________ 
2X SB 0.05M Tris-HCl 
1.0 
1% SDS 
10% Glycerol 
0.01% Brom. bl. 
2.5M NaCl 1.0M 0.8 
2-mercaptoethanol 
4% 0.08 
MilliQ H.sub.2 O 0.12 
Total ml = 2.0 
______________________________________ 
Collagen extractability was measured by cutting and weighing a sample piece 
from each tissue type with average size being 4.times.8 mm. An untreated 
tissue sample was included as a control. Each tissue sample was rinsed in 
approx. 1 mL MilliQ-UF water in individual microcentrifuge tubes for 5-10 
minutes and maintained on ice. The tissue pieces were patted dry with 
lint-free Kimwipes and weighed on an analytical balance (weight 
range=10-20.+-.0.2 mg; no less than 8). The pieces again were transferred 
to clean, individual microcentrifuge tubes and kept on ice. 
Two water baths were set up, one at 65.degree. C. and one boiling. A 
portion of extraction buffer (0.5M acetic acid) was added to each 
microcentrifuge tube, maintaining a constant concentration of 160 mg 
tissue per ml of buffer. E.g., a 16 mg piece of tissue would receive an 
aliquot of 0.10.+-.0.001 ml extraction buffer. The reaction tubes were 
tightly capped and placed in the 65.degree. C. water bath for 20.+-.0.25 
minutes The extraction was stopped by placing all tubes directly on ice.] 
Equal aliquots of each sample (e.g. 30 ul per sample per gel lane) were 
removed and added to 30 uL of the 2x sample buffer in a separate 1.5 mL 
microcentrifuge tube. These tubes were tightly capped and placed in a 
floating tube rack in the boiling water bath for 3-5 minutes. The samples 
then were chilled on ice and loaded onto a gel and electrophoresed for 350 
mA.hrs according to the methods described in PROTEAN II Dual Slab Cell 
Instruction Manual (Biorad Laboratories, Richmond, Calif.), incorporated 
herein by reference, using a 5-20% acrylamide gradient gel. The gel then 
was placed in a protein staining solution for approx. 1 hour and then 
destained until the colored background was clear. 
Results: 
Table 2 reflects the results of thickness measurement, shrink testing 
(T.sub.s), CNBr digestion, and collagen extract assay testing for each 
sample: 
TABLE 2 
__________________________________________________________________________ 
Bovine Leaflets Porcine Wrap 
Thickness 
Ts -Ave Extract 
Ts -Ave Extract 
pH 
Sample 
(% Change) 
of 2 CNBr 
Assay 
of 2 CNBr 
Assay 
&gt;Inc* 
__________________________________________________________________________ 
10 -1.01% 66.8 P** 
P 67.7 P P 6.60 
11 +0.46% 65.3 P P 64.8 P P 6.40 
12 -1.14% 66.0 P P 66.7 P P 6.42 
13 -0.22% -- P P -- P P 6.40 
14 +0.21% -- P P -- P P 6.40 
15 -0.79% -- P P -- P P 6.41 
19 +0.25% 65.3 P P 65.9 P P -- 
20 -0.42% 64.8 P P 65.4 P P 6.11 
21 -0.24% 64.7 P P 65.3 P P -- 
22 0.23% 63.5 P P 64.2 P P 6.08 
23 +0.53% -- P P -- P P 6.09 
24 0.00% -- P P -- P P 6.08 
25 -0.21% -- P P -- P P 6.08 
29 +0.21% 65.2 P P 64.3 P P -- 
__________________________________________________________________________ 
*pH &gt; Inc = pH after incubation. 
**P = Passed test, i.e. exhibited lack of digested or extractable 
proteins. 
Condition 1 solutions, whose sample number begins with a 1, initially had a 
pH of 6.5. Condition 2 solutions, whose sample number begins with 2, 
initially had a pH of 6.0. 
The noted change in thickness is quite small and approximates experimental 
error for this technique. The shrink temperature values (Ts) indicate a 
drop of 1.degree.-3.degree. C. from controls (nonincubated leaflets) run 
in parallel; however, all shrink temperatures remain above 62.degree. C. 
Therefore, the change is within acceptable limits. A P indicates a 
relative lack of detectable protein obtained through CNBr digestion when 
compared to digestion of nonphotooxidized tissue. Additionally, 
pretreatment of tissue with 2-mercaptoethanol prior to CNBr digestion 
resulted in tissue digestibility as seen with photooxidized controls. The 
collagen extraction assay resulted in a small quantity of unidentified 
lower molecular weight material, which was more prevalent with Condition 2 
treated tissue. The final pH of all samples was within 0.1 pH units of the 
original solution, and therefore was acceptable. 
Table 3 reflects the results of testing for microbial content. All of the 
samples listed, except 1C- and 2C-, were inoculated with 1.times.10.sup.7 
B. subtilis spores prior to the sterilization cycle. The components then 
were transferred to 50% ethanol. After one day in ethanol, the components 
were transferred to growth media and incubated for seven days to note any 
growth, indicating the presence of microorganisms. The iodine solutions 
and ethanol were filtered separately, and the filters were tested for 
microorganisms by submersion in growth media. In addition, a control media 
sample was inoculated to verify its ability to support microbial growth. A 
positive (+) indicates the presence of growth after seven days incubation 
while a negative (-) indicates no growth: 
TABLE 3 
______________________________________ 
Sample Components Filtered Iodine 
Filtered EtOH 
______________________________________ 
16 - - - 
17 - - - 
18 - - - 
1C+ - - - 
1C- - - - 
26 + + + 
27 + + + 
28 + + + 
2C+ - - - 
2C- - - - 
______________________________________ 
ADDITIONAL MICROBIAL KILL TESTS 
Experiment 9 
The purpose of the following test was to compare the ability of two 
concentrations of iodine solution to kill spores of Bacillus subtilis. Of 
the bacteria used in microbial kill tests using chemical sterilants, B. 
subtilis is one of the most difficult to kill. 
The results indicate that a 0.01% iodine concentration (Condition 2) was 
ineffective as a sterilant against spores of B. subtilis under the 
conditions of this test. Spores of B. subtilis survived in the 0.01% 
iodine solutions when incubated at 42.degree. C. in the presence of the 
components of the final device. Growth of the inoculated organism occurred 
in all three jars tested under this condition. No growth was detected from 
a container of iodine that was inoculated with spores but without tissue 
valve components. 
Although some Growth was noted in the B. subtilis samples, this does not 
mean that the solution failed to achieve a required kill rate. B. subtilis 
is one of the most difficult microorganisms to kill, and the B. subtilis 
samples were inoculated with 10.sup.7 spores. It should not be necessary 
to kill 10.sup.7 B. subtilis spores in order to pass regulatory standards. 
Incubation with the higher concentration solution of iodine (0.1%, 
Condition 1) at an incubation temperature of 37.degree. C. was effective 
to inactivate 10.sup.7 spores of B. subtilis per mL. All three jars tested 
using the 0.1% solution were sterile. The iodine solution alone, without 
components, also was sterile. 
All of the media that was used in this study was shown to support growth of 
B. subtilis, the negative controls for growth were negative and the 
microbial air sample was negative for growth in the sterility laboratory. 
Of the organisms detected on a microbial air sample in the general 
laboratory, no evidence of B. subtilis was found. 
It has previously been reported that the presence of organic matter may 
have an effect on iodine disinfection rates. For example, a 1:200,000 
(0.0005%) concentration of iodine reportedly will kill wet spores in the 
absence of organic matter in 15 minutes. From the following study (and 
from Table 3), it appears that the presence of the organic tissue valve 
components do tend to lower the disinfecting activity of these iodine 
solutions, as previously reported. 
Experimental Procedure 
Components of a pericardial valve were incubated for 48 hours.+-.2 hours, 
in the following two iodine solutions at different temperatures: 
______________________________________ 
Condition 1 Condition 2 
______________________________________ 
0.1% (w:v) I.sub.2 
0.01% (w:v) I.sub.2 
0.1% (w:v) NaI 0.01% (w:V) NaI 
0.1% (w:v) KI 0.01% (w:v) KI 
1X PBS 1X PBS 
.sup..about. 2% EtOH (from iodine stock) 
.sup..about. 2% EtOH (same) 
pH 6.5 pH 6.0 
incubation T.degree. = 37 +/- 2.degree. C. 
incubation T.degree. = 42 +/- 2.degree. C. 
______________________________________ 
Both iodine solutions were filtered with Nalgene Disposable Filterware, Lot 
#B10015, pore size 0.20u (cellulose nitrate filter). The excess ethanol 
was allowed to drip off of the components before placing them into the 
iodine solutions. Each jar was swirled by hand several times after closing 
the lid on the jar to be incubated. 
Prior to incubation, both iodine solutions were placed in a laminar flow 
hood and inoculated with 0.1 mL (10.sup.7) B. subtilis var niger spores 
obtained from North American Scientific Associates, Irvine, Calif., 
Lot#N16511, exp. May '92, 1.6.times.10.sup.7 spores/0.1 ml. The spore 
suspension was vortexed for approximately 5 minutes and the suspension was 
viewed under 100.times.for evidence of clumping. No clumps were observed. 
The spore suspension was then verified. 0.1 mL of suspension was added to 
9.9 mL of sterile water for injection to make 10.sup.6 spores/mL dilution. 
1.0 mL of 10.sup.6 spores was added to 9.0 mL sterile water to get 
10.sup.5 spores/mL. Further dilutions were made in a similar manner, to 
get 10.sup.4, 10.sup.3, 10.sup.2, 10.sup.1 spore concentrations using 
sterile test tubes and pipettes. 1.0 mL of 10.sup.2 spore dilution was 
spread onto 3 TSA plates. 1.0 mL of 10.sup.1 spore dilution was spread 
onto 3 TSA pates. All plates were incubated at 37.degree..+-.1.degree. C. 
for 48 hours. The number of B. subtilis colonies per mL were counted and 
the population count verified at an average of 1.3.times.10.sup.7 
spores/mL. 
After inoculation, all jars were torqued closed to approximately 32-34 
inch-pounds. The components then were transferred to approx. 80 mL of 50% 
ethanol which had been filter sterilized in the same manner as the iodine 
solutions and sterility tested. Sterility testing was performed by 
filtering through 0.45 u filter, rinsing with 100 mL of peptamin, and 
placing into 200 ml T-Soy Broth. No growth was observed after 7 days 
incubation at 35.degree..+-.2.degree. C. 
After 24 hours in the 50% ethanol, the components, the iodine solutions, 
and the ethanol solutions were tested for sterility. The contents of each 
jar was filtered through a 0.45 u filter unit and placed into approx. 200 
mL of T-Soy Broth and incubated at 35.degree..+-.2.degree. for 7 days and 
observed for growth. During in the hood sterility tests, a jar of media 
was left open continuously and closed upon completion of the test and 
incubated along with the other 3ars. After 3 days of incubation, no growth 
was observed in the media. Similarly, the forceps were dipped into 10 mL 
of T-Soy Broth which also was incubated at the same conditions. No growth 
was observed after 3 days. 
Media Preparation 
The solutions used in the procedure were prepared by the following methods: 
0.1% PEPTONE WATER 
1.0 g of peptamin (Bacto-Peptamin, USP XVII, DIFCO, Lot No. 796032) was 
mixed into 1.0 liter of distilled water. After mixing, the solution was 
dispensed in 100 ml quantities into milk dilution bottles and sterilized. 
Control: One bottle from each sterilizer load was left at room temperature 
for 24 hours. No growth was observed from the Peptone Water. 
TRYPTIC SOY BROTH (TSB) 
30 g of dehydrated DIFCO Dehydrated Tryptic Soy Broth, Lot No. 801810 (exp. 
10/96), was dissolved into 1.0 liter of distilled water. Approximately 200 
ml of media was dispensed into pint mason jars and sterilized. Media was 
prepared and sterilized as per USP XXII Section 71, incorporated herein by 
reference. The pH of the media was 7.25. 
Controls: 
(A) A Growth Promotion Test was performed to show that the media was 
capable of supporting Growth of a low number of microorganisms. This test 
was done by inoculating&lt;100 B. subtilis spores into one Jar of TSB. The 
medium was incubated at 35.degree. C. B. subtilis Growth was confirmed on 
day three. 
(B) All media was incubated for 24-48 hours at 35.degree. C. No Growth was 
observed indicating the sterility of the media before the test began. 
(C) Four jars of media were randomly chosen and incubated at 35.degree. C. 
for the duration of the test. No Growth reconfirmed the sterility of the 
media. 
TRYPTIC SOY AGAR (TSA) 20 g of DIFCO Dehydrated Tryptic Soy Agar, Lot No. 
795207 (exp. 95), was mixed with 500 ml of distilled water, and steam 
sterilized. Within the biological safety hood, the media was poured into 
sterile 100.times.15 mm petri plates. 
Control: B. subtilis was streaked onto one plate and incubated at 
35.degree. C. Growth on plate confirmed that the agar was capable of 
supporting growth. 
Sterilization/Incubation 
Solutions 
Solution 1 and Solution 2 were prepared as previously described. The iodine 
solutions were filter sterilized through a 0.2 .mu.filter unit. All white 
polypropylene jars used in this study were steam sterilized at 121.degree. 
C. for 30 minutes. Each transfer was performed individually. First, the 
jar was filled with approximately 80 ml of the iodine solution. Then the 
components were aseptically transferred to the iodine solution. The lid 
was tightened. Once the transfers were complete, each jar (#16-18 and 
#26-28) was inoculated with 10.sup.7 B. subtilis spores. The inoculation 
was conducted outside the sterility testing laboratory within the 
biological safety cabinet. Jars were torqued to approximately 32-34 inch 
pounds. Jars 16-18 were incubated for 48.+-.2 hours at 
37.degree..+-.2.degree. C. Jars 26-28 were incubated for 48.+-.2 hours at 
42.degree..+-.2.degree. C. 
Controls 
Before use, the spore suspension was vortexed and viewed under a microscope 
for evidence of clumping. The spore population was verified by preparing 
serial dilutions to a 10.sup.1 spores/ml dilution and plating 1.0 ml onto 
triplicate TSA plates. The plates were incubated at 
37.degree..+-.2.degree. C. for 48 hours. The number of colony forming 
units were counted, and the initial spore concentration was verified, as 
shown in Table 4: 
TABLE 4 
______________________________________ 
Dilution CFU/ml No. spores/0.1 ml 
______________________________________ 
1:10.sup.6 140 1.4 .times. 10.sup.7 
1:10.sup.7 12 1.2 .times. 10.sup.7 
______________________________________ 
average = 1.3 .times. 10.sup.7 spores/0.1 ml 
labelled = 1.2 .times. 10.sup.7 spores/0.1 ml 
Since the average spore population was determined to be 1.3.times.10.sup.7 
spores/0.1 ml and the labelled spore population was 1.6.times.10.sup.7 
spores/0.1 ml, the spore population was confirmed to be 10.sup.7 
spores/0.1 ml. 
The temperature of the incubators was continually monitored to assure 
constant temperatures throughout the incubation period. The temperature 
range of the 37.degree. C. incubator was 37.3.degree. C. to 37.4.degree. 
C. The temperature range of the 42.degree. C. incubator was 41.6.degree. 
C. to 42.1.degree. C. The sterility lab was cleaned and particle counts 
were taken to assure that microbial contamination for the sterility test 
laboratory was within 10,000 particles per cubic foot at 0.5 microns and 
less than 70 particles per cubic foot at 5.0 microns, and within the 
laminar flow hood less than 100 particles per cubic foot at 0.5 microns. 
Transfer to 50% EtOH 
The 50% EtOH was filter sterilized through a 0.2 .mu.filter unit. Each 
transfer was performed individually. First, approximately 80 ml of the 50% 
ethanol was poured into a white plastic sterile jar. The components were 
transferred from the iodine solutions to the 50% EtOH using sterile 
forceps. The transfer of jars inoculated with B. subtilis were made 
outside the sterility testing laboratory within the biological safety 
hood. During the transfer, observations of the tissue, solution condition, 
and color were made and recorded. Table 5 below outlines these 
observations. All jars sat at room temperature for approximately 24 hours 
before conducting the sterility test. 
Controls: The filtered 50% EtOH was tested for sterility (as described 
below in Sterility Testing). Two TSA plates were left open in the hood 
during the transfer, and then incubated at 35.degree. C. for 48 hours. 
During the transfers, microbial air sampling was performed within the 
sterility testing laboratory and in the microbiology laboratory. 
Sterility Testing of Components 
After approximately 24 hours in the 50% EtOH, the components of each jar 
#16-18 and #26-28 were tested for sterility. Individually, the contents 
from each jar were transferred to 200 ml of TSB. The broth was incubated 
for 7 days at 35.degree..+-.2.degree. C. Each day the media was examined 
for growth. 
Sterility Testing of Iodine/Ethanol Solutions 
Individually, each solution from each jar #16-18 and #26-28 was filtered 
through a 0.45 .mu.filter on a 3-place filter holder manifold. Testing was 
conducted in the biological safety hood in the general microbiology 
laboratory area. Each filter was rinsed three (3) times with 100 ml of 
peptamin. The filter was placed into 200 ml of TSB and incubated at 
35.degree..+-.2.degree. C. for 7 days. Each day the media was examined for 
growth. 
Controls: During the sterility test in the biological safety cabinet, one 
jar of medium was left open continuously. Upon completion of the tests the 
jar was closed and incubated with the others. One forceps was dipped into 
10 ml of TSB then incubated at 35.degree. C. One filter was rinsed three 
(3) times with 100 ml of peptamin, placed into 200 mil of TSB, incubated 
and observed for growth. 
Observations 
Table 5 outlines the tissue condition, tissue color, and iodine solution 
color after incubation in iodine solutions: 
TABLE 5 
______________________________________ 
Tissue Iodine 
Jar Conditions 
Tissue Color Soln Color 
______________________________________ 
10 fresh, 2 leaflets-white, 2 leaflets-629C* 
(no I.sub.2) 
pliable 
11 fresh, tissue = 557C*, components = off 
580C* 
pliable white (yellowish) 
12 fresh, tissue = 579C*, components = off 
" 
pliable white (yellowish) 
13 fresh, tissue = 579C*, components = off 
" 
pliable white (yellowish) 
14 fresh, tissue = 557C*, components = off 
" 
pliable white (yellowish) 
15 fresh, tissue = 557C*, components = off 
" 
pliable white (yellowish) 
16 fresh, tissue = 557C*, components = off 
" 
pliable white (yellowish) 
17 fresh, tissue = 564C*, components = off 
395C* 
pliable white (yellowish) 
18 fresh, tissue = 564C*, components = off 
387C* 
pliable white (yellowish) 
19 fresh, tissue = 629C*, components = no 
-- 
pliable discoloration (ethanol) 
20 fresh, tissue = 630C*, components = no 
636C* 
pliable discoloration (no iodine) 
21 fresh, tissue = 630C*, components = no 
636C* 
pliable discoloration (no iodine) 
22 fresh, tissue = 630C*, components = no 
629C* 
pliable discoloration 
23 fresh, tissue = 629C*, components = no 
636C* 
pliable discoloration 
24 fresh, tissue = 629C*, components = no 
628C* 
pliable discoloration 
25 fresh, tissue = 629C*, components = no 
" 
pliable discoloration 
26 fresh, tissue = 629C*, components = no 
" 
pliable discoloration 
27 fresh, tissue = 629C*, components = no 
635* 
pliable discoloration 
28 fresh, tissue = 629C*, components = no 
628* 
pliable discoloration 
29 fresh, -- -- 
pliable 
______________________________________ 
*Color matches the numbering system of the Pantone Color Chart 1991. 
From the foregoing results, it was concluded that tissue treated with 
iodine sterilization solutions may exhibit slight staining; however, any 
staining appeared to be temporary only, and, in fact, appeared to be 
reversed by subsequent soaking in 50% ethanol. 
Results From Controls 
Table 6 shows the result of the controls mentioned in the procedure. All of 
the controls were negative for growth and thus sterile. 
TABLE 6 
______________________________________ 
Test Controls Results 
______________________________________ 
Open TSA Plates Negative 
Forcep Negative 
System Control Negative 
Ethanol Negative 
Negative Control Negative 
Air Sampling 
Sterility Lab No growth 
General Lab 4 CFU/ft.sup.3, no B. subtilis 
______________________________________ 
Sterility Test Results 
Condition 1 
As seen in Table 7, the tissue valve components, the iodine solution, and 
the ethanol which was exposed to Condition 1 all were sterile for jars 16 
to 18. The inoculated iodine without components (jar C1+) also was 
sterile. 
TABLE 7 
______________________________________ 
Sample Results 
______________________________________ 
jar 16 components Sterile* 
jar 17 components Sterile 
jar 18 components Sterile 
jar 16 iodine solution Sterile 
jar 17 iodine solution Sterile 
jar 18 iodine solution Sterile 
jar 16 EtOH Sterile 
jar 17 EtOH Sterile 
jar 18 EtOH Sterile 
jar C1- Sterile 
jar C1+ Sterile 
______________________________________ 
*Sterile = No growth was detected 
Condition 2 
As seen in Table 8, the tissue valve components, the iodine solution, and 
the ethanol from Condition 2 all were non-sterile. Colony morphology and 
gram staining confirmed that all non-sterile jars contained B. subtilis. 
The inoculated iodine solution without components from Condition 2 (C2+) 
was sterile. All jars of media which were positive for growth were 
streaked onto a TSA plate and incubated at 35.degree. C. All growth was 
confirmed to be B. subtilis by colony morphology and a gram stain. 
TABLE 8 
______________________________________ 
Sample Results 
______________________________________ 
Jar 26 Components Non-Sterile 
Jar 27 Components Non-Sterile 
Jar 28 Components Non-Sterile 
Jar 26 Iodine Solution 
Non-Sterile 
Jar 27 Iodine Solution 
Non-Sterile 
Jar 28 Iodine Solution 
Non-Sterile 
Jar 26 EtOH Non-Sterile 
Jar 27 EtOH Non-Sterile 
Jar 28 EtOH Non-Sterile 
Jar C2- (un-inoculated) 
Sterile 
Jar C2+ (inoculated) Sterile 
______________________________________ 
Experiment 10 
The purpose of this experiment was to challenge a candidate sterilant 
solution (0.1% iodine solution buffered to pH 6.5) with Pseudomonas 
aeruginosa, Bacillus subtilis, and Staphylococcus aureus. 
The samples were inoculated at approximately 1.times.10.sup.6 per jar, 
1.times.10.sup.7 per jar for B. subtilis, with the following organisms: 
B. subtilis, spore suspension, NAmSA; 
P. aeruginosa, ATCC 9027; 
S. aureus, ATCC 6538. 
The candidate sterilant contained 0.1% iodine phosphate buffered to pH 6.5 
(preparation already described), and the incubation lasted for 48.+-.2 
hours at 37.degree..+-.2.degree. C. The following test samples were used: 
Samples innoculated with B. subtilis: 
1 (a-c): no tissue valve components--3 jars; 
1 (d-h): with tissue valve components--5 jars; 
Samples innoculated with P. aeruginosa: 
2 (a-c): no tissue valve components--3 jars; 
2 (d-h): with tissue valve components--5 jars; 
Samples innoculated with S. aureus: 
3 (a-c): no tissue valve components--3 jars; 
3 (d-h): with tissue valve components--5 jars; 
Samples not innoculated: 
CI: iodine only, no tissue valve components--4 jars; 
CIC: with tissue valve components--1 jar; 
CE: ethanol solution--4 jars. 
Unless otherwise stated, all tissue was photooxidatively stabilized. The 
color and pH of the iodine solution from jar CIC was noted, and the 
appearance before and after transfer into 50% ethanol was noted. 
The following results were obtained in the following samples, each of which 
contained tissue valve components in addition to the iodine and inoculum: 
No growth in five samples with 10.sup.6 Escherichia coli; 
No growth in five samples with 10.sup.6 Staphylococcus aureus; 
Growth in 1 of 5 samples with 10.sup.7 Bacillus subtilis. 
Control samples inoculated in the absence of tissue valve components all 
indicated no surviving microorganisms. 
Although some growth was noted in the B. subtilis samples, this does not 
mean that the solution failed to achieve a required kill rate. B. subtilis 
is one of the most difficult microorganisms to kill, and the B. subtilis 
samples were inoculated with 10.sup.7 spores. It should not be necessary 
to kill 10.sup.7 B. subtilis spores in order to pass regulatory standards. 
Experiment 11 
Experiment 10 was repeated using Escherichia coli, Candida albicans, and 
Bacillus subtilis. The samples again contained tissue valve components in 
addition to the iodine solution and inoculum. Similar results were 
obtained and given a similar interpretation: 
No growth in five samples with 10.sup.6 E. coli; 
No growth in five samples with 10.sup.6 C. albicans; 
Growth in 2 of 5 samples with 10.sup.7 B. subtilis. 
Control samples (3 for each microorganism) inoculated in the absence of 
tissue valve components all indicated no surviving microorganisms. 
Experiment 12 
The following experiment was designed to examine the effect that refreshing 
the sterilization solution at intervals would have on microbial kill by 
various solutions. Samples were prepared to contain all tissue valve 
components, 80 mL iodine sterilants (at 0.01, 0.02, 0.05, or 0.10% 
iodine--six samples at each concentration), and 1.35.times.10.sup.6 
Bacillus subtilis. Controls contained no components. At 24 hr (one-half of 
the sterilization cycle), components (when present) were transferred to a 
fresh polypropylene jar with fresh iodine. To four samples (at each 
concentration) was added a filter obtained from the filtration of the 
iodine solution from that sample. This was done to carry over any 
surviving organisms. 
The results are reflected in the following chart, in which the headings 
have the following meanings: 
Sample--sample number 
Iodine--concentration (each) of iodine, KI, and NaI 
Components--presence or absence of tissue valve components 
Filter added back--all iodine samples were filtered at 24 hours after 
component transfer to fresh iodine. In four samples at each iodine 
concentration (e.g., 1A-1D) the filters were tested for sterility. In the 
four other samples (e.g., 1A'-ID'), the filters were added to the new 
iodine. 
Filter sterile? (24 hour)--The filters not added to the new iodine were 
tested for sterility. Y indicates no Growth. 
Final solution sterile--After the total 48 hour sterilization cycle, 
solution was tested for sterility. 
TABLE 9 
______________________________________ 
Filter *Final 
Iodine Com- Filter Sterile? 
Solution 
Sample Conc. ponents Added Back 
(24 hr.) 
Sterile? 
______________________________________ 
1A/1A' 0.01 - N/Y Y Y/Y 
1B 0.01 + N N N 
1C 0.01 + N N N 
1D 0.01 + N N N 
1B' 0.01 + Y NA N 
1C' 0.01 + Y NA N 
1D' 0.01 + Y NA Y 
2A/2A' 0.02 - N/Y Y Y/Y 
2B 0.02 + N N N 
2C 0.02 + N N N 
2D 0.02 + N N N 
2B' 0.02 + Y NA N 
2C' 0.02 + Y NA N 
2D' 0.02 + Y NA N 
3A/3A' 0.05 - N/Y Y Y/Y 
3B 0.05 + N Y Y 
3C 0.05 + N N Y 
3D 0.05 + N N Y 
3B' 0.05 + Y NA Y 
3C' 0.05 + Y NA Y 
3D' 0.05 + Y NA Y 
4A/4A' 0.10 - N/Y Y Y/Y 
4B 0.10 + N Y Y 
4C 0.10 + N Y Y 
4D 0.10 + N Y Y 
4B' 0.10 + Y NA Y 
4C' 0.10 + Y NA Y 
4D' 0.10 + Y NA Y 
______________________________________ 
*Final solution tested for sterility for samples without/with filter adde 
back (this applies only to the no component samples). 
The foregoing results indicate that changing the iodine based solution 
during the cycle--here at mid-point--may increase the efficacy of the 
solution in killing microorganisms. 
Experiment 13 
The following experiment was conducted to determine whether or not a 
solution of pure elemental iodine would be capable of safely and 
effectively sterilizing the tissue implants. 
Experimental conditions 
80 mL 0.1% iodine (with and without each of KI and NaI) 
pH 6.5, lX PBS 
2% total ethanol 
4.times.1" squares bovine photofixed tissue 
1.35.times.10.sup.6 B. subtilis spores 
Incubation for 48 hours at 37.degree. C. 
After incubation, sterility tests were performed on tissue samples, iodine 
sterilant sample, and filter obtained by filtering the remainder of the 
sterilant. The following results were obtained. 
TABLE 10 
______________________________________ 
Sterility results 
Tissue Solution 
Filter 
Sterilant Cotents Sterile? Sterile? 
Sterile? 
______________________________________ 
iodine tissue + 3/3-Yes 3/3-Yes 1/3-Yes 
only spores 2/3-No 
iodine tissue NA NA NA 
only only 
iodine spores 3/3-Yes 3/3-Yes 3/3-Yes 
only only 
+iodides tissue + 3/3-Yes 3/3-Yes 3/3-Yes 
spores 
" tissue NA NA NA 
only 
" spores 3/3-Yes 3/3-Yes 3/3-Yes 
only 
______________________________________ 
TABLE 11 
______________________________________ 
Shrink temperature results 
Sample A B C 
______________________________________ 
Iodine with no 
65 64.6 65.1 
iodides (0.2) (0.4) (0.1) 
Iodine with 63.9 59.6 59.7 
NaI & KI (1.3) (2.4) (2.3) 
______________________________________ 
(Numbers in parentheses are degrees below control which is a photooxidize 
and nonsterilized bovine leaflet. Controls are included in each shrink 
temperature run.) 
From these results, it was concluded that: 
1) An iodine solution, without stabilizing salts, is capable sterilizing 
tissue implants; and 
2) The efficacy of an elemental iodide solution without stabilizing salts 
is less than the efficacy of a solution with such salts. Therefore, it is 
possible that the stabilizing salts maintain the iodine in an active 
(antimicrobial) form. 
IMPLANTATION STUDIES 
Experiment 14 
In order to assess the efficacy of the present sterilization method in 
vivo, tissue valves that had been fixed and sterilized using 
glutaraldehyde were compared to tissue valves that had been fixed and 
sterilized according to the methods described herein. The implanted valves 
included clinically available Carpentier-Edwards porcine valves and 
Carbomedics valves. The valves were implanted in sheep for a period 
varying between 26-157 days. The valves later were explanted and studied 
for evidence of calcification and other damage. 
Several of the animals in the study suffered early deaths which appeared to 
be due to infection and related thrombosis. A 10-20% infection rate is 
expected in animal related implant studies. One control valve failed early 
due to a leaflet tear, and another control valve failed early because of a 
size mismatch resulting in stent distortion. Two of the valves were 
treated with a higher temperature and lower pH than preferred, which is 
known to cause collagen denaturation, but at a relatively low pH. These 
two valves did not show any calcification, and performed satisfactorily 
with no degenerative problems. 
All of the glutaraldehyde treated valves exhibited some degree of 
calcification with short implant durations. The calcification can be seen 
at the coaption points in FIGS. 7A, 7B, and 7C and in FIGS. 8A and 8B, 
which are photographs of x-rays of representative explanted tissue valves 
treated with glutaraldehyde. All but one of the valves treated according 
to the methods described herein performed successfully through a five 
month implant time, did not show any calcification, and performed with no 
degenerative problems. The-other valve failed due to a non-valve related 
problem after two months, and also did not show any calcification. The 
absence of calcification can be seen in FIGS. 6A and 6B, which are 
photographs of x-rays of explanted tissue valve treated according to the 
present invention. 
Experiment 15 
Six valves (designated as "Zeta.sub.-- ") treated as described herein were 
implanted and explanted after the following time periods with the 
following results: 
The following samples gave a physiologic gradient and no regurgitation via 
angiography. The tissue was in good shape with good leaflet flexibility 
and no visual calcification. Zeta 25, explanted after 163 days; Zeta 26, 
explanted after 161 days; Zeta 27, explanted after 160 days; and Zeta 30, 
explanted after 154 days. 
Zeta 28, explanted after 159 days, demonstrated a higher than expected 
gradient and had significant regurgitation; however, upon valve retrieval, 
it was observed that sutures were looped over two stent posts which 
interfered with the functionality of the leaflets. The animal was in good 
health and the valve functioned adequately enough, even with the suture 
loops to keep the animal alive. The extra stress of the loops would be 
expected to result in rapid calcification and early animal death in a 
glutaraldehyde treated valve. 
Zeta 29, explanted after 155 days, had a good gradient but demonstrated 
slight regurge, as indicated by a wisp of dye backflowing after valve 
closure. The explant revealed one lazy leaflet which might have resulted 
in slight prolapse. This possibly is due to slightly different tissue 
stretch, leaflet-to-leaflet. Again, as in other explants, the valve was 
clean and the leaflets looked good. 
One of skill in the art will appreciate that many modifications may be made 
to the embodiments described herein and explained in the accompanying 
figures without departing from the spirit of the present invention. 
Accordingly, the embodiments described herein are illustrative only and 
are not intended to limit the scope of the present invention.