CA2169478C - Electron beam sterilization of biological tissues - Google Patents

Abstract

The invention involves a method for sterilizing biological tissues. The invention also includes sterilized biological tissues.

Description

- ~ _ 21 69478 WO95r236lG PCT~S95l0283 ELECTRON BEA~ STERILIZATION OF BIOLOGICAL TISSUES

Technical Field:
The invention involves sterilized biological tissue~
and methods for sterilizing biological tissues.

Bac~qround of the Invention Surgical i~plantation of tissue is utilized to replace and/or repair human tissues. Por example, ~ereditary defects, disease, andlor trauma may damage tissues such that replacement and/or repair is desirable.
These implantable tissues may be provided by individual ~u~an donors. However, because of the scarci~y of appropriate human donors, non-human tissues have been increasingly employed instead. 5uch biological tissues have been used to replace heart valves, ligaments, tendons and skin, a~ong other tissues.
Biological tissues deriYed from non-human or non-self sources may pose formidable problems to the new recipient. For example, the recipient's immune system may react to t~e implanted tissue and form an immune response, potentially leading to rejection of the implanted tissue. Thus, the new tissue ~ay become ineffective and/or exhibit poor durability once implanted.
Conventionally, glutaraldehyde has been used to addrecs so~e of the5e problems and to stabilize the tissue against in ~ivo enzymatic degradation.
Additionally, glutaraldehyde has been used as a sterilizing agent to inhibit the infectivity of implant tissue. Glutaraldehyde cross-links proteins rapidly and effectively, particularly proteins such as collagen.
This treatment increases resistance to proteolytic cleavaqe and hence increases resistance to enzymatic 35 -degradation.
In addition to crosslinking with glutaraldehyde, it is also well known to sterilize the crosslinked tissue wo ssn3cl6 PCTtU59i/n2g3 with gamma radiation and the like prior to storage of the biological tissue.
Ga~ma radiation, and similar sterilization pr~tocols, transfers energy to material primarily by Compton ccattering i.e., ~cattering involving elastic collisions between incident photons and unbound (or wea~ly bound) electrons in which the incident energy is shared between the scattered electron and the deflected photon. These electrons recoil a short distance as lo unbound electrons, giving up energy to the molecular structure of the material as they collide with other electrons, causing ionization and free-radical formation.
The scattered gamma ray carries the balance of the energy as it ~oves off through the material, possibly to interact again with another atomic electron. Since the probability for Compton scattering is low, gamma rays typically penetrate relatively deeply into the tissue before scattering occurs. Accordingly, gamma rays deposit energy in ~aterial over relatively large volumes so that penetration is typically high (typical~y greater than 50 cm in unit-density material) but dose rates are typically low (typically about a maximum of 20 ~Gy/hr).
See Figure 1.
Most techniques for sterilizing biological tissues produce undesirable results in the material, but the undesirable results may be more prominent when gamma radiation is used. Such undesirable results include but are not limited to the formation of radicals, hydrogen, and low-molec~lar-weight hydrocarbons; increased ~o unsaturation; discoloration; and oxidation. ~urthermore, gamma radiation typically requires a low dose rate in combination with a high exposure period, and degrades the structure of most conventional packaging materials.
Biological tissues prepared by the p~ior art methods suffer from a number of disadvantages, which li~its their use in implantation, particularly human implantation.
~irst, the use of some chemical sterilizing agents e.g.,

2 1 69478 Wo9~1~616 PcT~S95/0~3~

glutaraldehyde, increases the risk ~hat a toxic response will be evoXed in sensitive individuals, even after thorough rinsing of.the tissue prior to implantation.
Second, t~e use of certain sterilizing agents requires t~at t~e tissue be sterilized prior to packaging, thus necessitating a packaging step which must be carried out under stringent aseptic conditions. Third, gamma radiation can degrade polymeric materials employed in pac~aging by facilitating damaging oxidative reactions of polymers. Fourth, because gamma ~adiation typically involves relatively lo~ dose rates, correspondingly long periods of exposure to effec~ sterilization ~ay be necessary.
Thus there is an unaddressed need in the art for a method of sterilizing biological tissues that ~inimizes t~e possibility of immune rejection. Additionally, there exists a long-felt need ~or a method of sterilizing that does not necessitate an aseptic packaging step. Further, there is a need in the art for a ~ethod of sterilizing tissues which does not deg~ade the polymeric materials employed in packaging sterili2ed biological ti sues.
There i5 also a need for a method of sterilizin~
biological tissues that is quick, efficient, and ~esults in a biological tissue with enhanced performance characteristics.

Su~mary of the Invention The present invention encompasses sterilized biological tissues and ~ethods o~ sterilizing biological tissues which reduce or eliminate the disadvantages noted above.
In accordance with the present invention, biological tissues are treated by exposing the ticsue to E-beam radiation sufficient to effect sterilization.
Addi~ionally, t~e present invention provides a biological tissue sterilized ~y E-bea~ radiation, with the resulting biological tissue exhibiting enhanced perfor~ance - ~_ 2169478 wossr23616 PCT~S9~/0283 characteristics. The methods and tissues according to the present invention have the added advantage of reduced risk of infectivity, and eliminates the need for aseptic ~andling protocols. Further, the methods and tissues of S the present invention, which u~e fewer reagents and/or require less processing, provide for lower costs in labor, reagents, time and personnel. E-beam radiation sterilization is effective in obviating the need for toxic sterilizing che~icals. Moreover, the amount of radiation required for E-beam sterilization does not significantly degrade the biological tissue, t~us providing a more durable transplantable tissue.
~ here are a large number of characteristics that distinguish accelerated elect~ons from gamma rays:
Source of Radiation. Gam~a rays are e~itted by the decay of Co~alt-60. E-beams are produced by accelerating electron systems such as linear accelerators, Dynamitrons, and Van de Graa~f generators.
Dose Rate. The dose rate for gamma radiation is approximately llO grays per minute and the dose rate of E-beam is approximately ~800 grays per minute.
Consequently, exposure times are dramatically greater for 2~ ga~ma radiation, which requires low doses over an extended period to effect sterilization. In contradistinction to gamma r~diation, the high dose rates involved in E-beam irradiation promote diffusion of oxygen into biological tissue at a rate insufficient to par~cipate in free radical formation reactions, such 25 those which might contribute to tissue and polymer degradation. This is particularly advantageous in those embodiments which include placing the biological tissue in a container prior to irradiation, since polymer degradation in both the tissue and the ~ontainer may be minimi2ed.

~` 2169i78 w095/23616 ~CI~S9~l028 ~ rther~ore, the high dose rate of E-beams relative to gamma rays permits a higher processing rate of sterilization, commonly an order of magnitude higher.
For example, the sterilization period may be a matter of minutes for E-beam treatment, in contrast to the hours or ~ore for ga~ma radiation treatment. Yet ~espite the higher dose rate, the present process does not result in signific~nt degradation of biological tissue during ~he sterili~ation process.
Penetration. In relative terms, gamma radiation penetrates approximately ten times further into materials than 10 MeV electrons in the same material. Because the probability for electron-electron and electron-nuclear scattering may be high (relative to Compton scattering~, ~0 MeV E-beams typic~lly penetra~e approximately 5 cm in unit-density materia~ before losing t~eir energy. Thus, the po~er in the beam is typically deposited within a narrow range in the material and concentrated within the width of the beam. ~his results in high dose rate and low penetration (300 kGy per pulse, with an average dose rate of 2.2 x lo' kGylhr for a 50 kw beam; 5 cm depth).

Directionality. The material is bombarded with electrons from a single direction, whereas materials are exposed to ga~ma rays from all directions.

Uniformi~y of Exposu~e. A more uniform reaction is ac~ieved from gamma radiation than from E-beam radiation.
Limited and differential penetration through materials result in "shadowing" with E-beam.

Source of ~xcited Electrons. Gamma rays induce excitation of electrons within the atoms of the materials to be sterilized. Electron beams, on the other ~and, provide high-energy electrons to the exte~ior of the WO95123616 PCT~S93/0283 material, which in turn put subsequent electrons in ~otion.

Polymer Dose Res~onse. Several studies report differences in the response of polymers, particularly polyethylene and polypropylene, to gamma and E-~eam radiation. Because of the relatively high dose rate of the E-beams, oxygen is not capable of diffusing into the material ~t a rate required to participate in oxidati~e reactions that may lead to degradation of the material.
Furthermore, prevention of degradation in both the package and the tissue permit terminal sterilization, i.e., sterilization of the tissue in its final, sealed package. Thus, the present invention avoids the need for costly aseptic handling techniques, and provides sterility assurance as long as the package is intact, i.e., until the tissue is ready for use.

Micro-orqanism Dose ~esponse. Early studies by an independent agency suggest a difference in the response of certain bacteria to gamma and E-beam radiation.

Dose Build-up. The phenomenon called build-up occurs with electron beam radiation only. As high energy electrons penetrate the surface they collide with atomic electrons of t~e material. These electrons, in turn, recoil and collide to set more electrons in motion so that from a relatively few electrons penetrating the surface, there results a multiplicity of electrons depositing energy in t~e material, primarily by the production of ions and free radicals. ~his process, called ~uildup, resul~s in higher doses being delivered to depths below the surface where the primary beam and its recoil electrons can no longer produce ionization.
Thereafter, the electrons quickly lose their remaining energy, pri~arily by soft interactions with atomic electrons (excitation) and radiative losses.

2t 69478 wo 95/236~6 PCT~Sg~t0283 Precise and Reliable Dosimetry. E-beam dose can be carefully controlled because each product ~s irradiated individ~ally on a conveyor, a factor which may be very important when critical doses are required. On the other ~and, in a gamma facility many packages of a wide variety of materials are irradiated simultaneously. The dose delivered to each package may subsequently vary due to s~ielding effects caused by the density differences among the products.

Option of Differential Irradiation. With E-beam radiation, each package is irradiated individually with its own specified beam current, energy and exposure time.
Additionally, parts of a package may be irradiated at a different level than other parts of the same package.
For example, with E-beam radiation it is possible to irradiate half of a box with a dose of 10 kGy and the other half of the box with 25 kGy. It is also possible, using the limited penetration of E-beam to one's ad~antage, to shield sensiti~e parts of an object.

E~se and SafetY of Operation. Wi~h the levels of energy utilized in an E-beam facility, there is li~tle or no activation (induced radioactivi~y) of materials.
Therefore, when the accelerato~ is turned off there is no danger of radiation ~azard. Further~ore, there are no specific requirements for handling, monitoring, or disposing a radiation source.

30 Temperature Rise. An issue that must be carefully considered when using ionization radiation to sterilize collagen-based materials is that of heat generation during the irradiation process. The temperature rise per megarad (Mrad) of deposited energy tl Mrad = 10 kGy) is calculated by dividing the heat equivalent of a Mrad (2.39 calorieslgram) by the specific heat of the mate~ial. This formula applies to both E-beam and gamma W095/23616 PCT~S9~10283 radiation. For biological vascular graft tissue and packaging solutions (specific heat =l cal/g-C), temperature rise for a 2.5 Mrad dose is as follows (for a biological graft on a glass mandrel):
S

2.39 calories/gram x 2.5 = 6.0C
l.0 calories/gram-C

For a vascular graft on polycarbonate mandrel (specific heat - 0.28 cal/g-C) a greater rise in tempera~ure can be expected:

2.39 calories/qram x 2.5 = 21.3C
0.28 calories/gram-C
Given that the ambient t~ ,?rature of a typical E-beam facility is approxi~ately 20C, the maximu~
theoretical final temperature of ~he polycarbonate mandrel would be approximately 41C. The ambient temperature of a typical ga~ma cell facility on the o~her hand, is in excess o~ 38C. The final temperature therefore, could reach approximately 59C. ~urthermore, an exposure ~ime at this temperature would be a number of hours, as opposed to a few minutes ~ith E-beam.
Brief DescriPtion of the Figures Figure l Interaction of Gamma Rays and Electron Beams with Material.
Figure 2 Incre~se in Dose With Depth of E-Beam Penetration. ' Figure 3 Position vs. ~-Signal.
Figure 4 Therascan Contour Depiction of BPMrM Graft.
~igure 5 Bovine Pericardium Shrink Temperature - 0.01%
Glutaraldehyde5 Figure 6 0.1% Glutaraldehyde - Fixed Bovine Carotid Artery; Shrink Te~perature and Glutaraldehyde Depletion.

wossn3616 PCT~S9S/02835 Figure 7 Orientation of Electron Beam Scanner and Conveyor.
Figure 8 Shows the effects of E-~eam radiation on pressure drop.
Figure 9 Shows the effects of E-beam on effective orifice area.

SPecific Descri~tion of the Invention The present invention includes a method for sterilizing a biological tissue comprising directly exposing the tissue with z bea~ of accelera~ed electrons to sterilize the tissue. In a preferred em~odiment, the sterilization is terminal sterilization.
The present invention als~ ~ncludes a method for ~5 sterilizing a biological tissue co~prising irradiating a crosslinked tissue in a container with a beam of accelerated electrons to sterilize the tissue and container. In a preferred embodi~ent, the container is sealed prior to sterilization and the tissue and container are subjected to terminal sterilization.
The present invention also includes a sterilized biological tissue comprising a biological tissue treated according ~o a ~et~od of the invention.
The present invention also includes a sterilized biological tissue having improved performance characteristics.
The term '1biological tissue" as used herein refers to a collagen-containi~g material ~hich may be derived from different animal species, typi~ally mam~alian. The biological tissue is typically a soft tissue suitable for implan~ation, such as bioprost~etic tissue or the like, but the invention s~ould not be limited thereby.
Specific examples include, but are not limited to, heart valves, particularly porcine heart valves; aortic roots, walls, and/or leaflets; pericardiu~, preferably bovine pericardium or the like, and produc~s derived from pericardium, such as a pericardial patch; connective W095/23616 PCT~S~StO28 tissue derived materials such as dura mater; homograft tissues, such as aortic homografts and saphenous bypass grafts; tendons, ligaments, skin patches; blood vessels, particularly bovine arteries and veins, and human u~bilical tissue, such as veins; bone; and t~e like. Any other biologically-derived ~aterials which are known, or become known, as being suitable for processing in accordance with the invention are within the contemplation of the invention.
In accordance with tbe invention, the biological tissue, explanted from its source ~ay be processed in any suitable manner prior to exposure to a crosslinking agent.
The term "sterilization" as used herein refers to exposing the biological tissue to a sterilizing beam of accelerated electrons, i.e., an ~-bea~. The particle beam which comprises the E-bea~ preferably includes directional bombardment, i.e., bombardment from one direction only, and includes single-side or multiple-side irradiation.
The beam of accelerated particles may be provided by an electron scanner or accelerator (10) capable of generating beams (12) with energies of, for example, 10 mega electron-volts tMeV). See ~igure 7. As noted above, one skilled in the art ~ill recognize that the energy of the beam effects only the depth of penetration, not the exposure time, and selecting the appropriate energy setting is dependent, in par~, on the dimensions of t~e specific package or object.
In accordance with the invention, the amount of E-beam radiation is an amount sufficient to sterilize ~he biological tissue, and in some embodiments, an amount sufficient to sterilize the biological tissue packaged in its final container. One skilled in t~e art will recognize and be able to determine a sterilizing dose and time suitable for a particular tissue and based on the characteristics of the accelerator being used.

~ 2 1 6q47~

~095123616 PCT~S9~/0283 Typically, the biological tissue is subjected to a one-sided exposure to the electron beam until a sterilizing dose of radiation is absorbed. Absorbed dose of radiation is expressed in terms of kilograys (kGy).
One kilogray is equal to one thousand joules of energy deposited per kilogram of material. For example, the biological tissue may be irradiated until a dose o~
approximately 25 kGy or more is achieved. ~or example, the FDA presently requires a dose of 25 kGy or greater for the sterilization of medical products. For the present invention, while upper and lower limits on the sterilizing dose have not yet been determined, sterilizing doses of greater than 25 kGy haYe ~een found effective, typically from about 25 kGy to about 28 kGy.
In a preferred ~hoAiment of the invention, the biological materia} is subjected to a one-sided top exposure to an electron beam until a top surface dose of approxi~ately 25 to 28 kGy is achieved.
Irradiation may be carried out in a conventional manner, i.e. by placing the biological tissue in a suitable container, e.g., a glass or plastic container, and exposing the tissue to the electrons. ~or example, the biological tissue (16).may be placed in an aluminum tote (18) on a conveyor t14) which then passes throug~
the electron beam (12). See Figure 7. Typically, the time of exposure to the beam ~ay be proportional to the dimensions of the biological tissue~ For example, a single row o~ heart valves (approxi~ately eight valves) can be irradiated in approximately one minute, based upon a conveyor speed of 0.l c~ per second and a valve jar 5.5 cm in diameter.
Effective sterilization may be easily determined using conventional micro~iological techniques, such as for example, the inclusion of suitable biological ~5 indicators in the radiation batch or contacting the tissue with a culture mediu~ and incubating the mediu~ to determine sterility of t~e tissue. Dose may also be .` _ 2 1 6q478 WO95123616 PCT~S9510283 determined with the use of radiochromic dye films. Suc~
films are calibrated, usually in a gamma field, by reference to a national standard.
Degradation of the biological tissue by irradiation may also be determined using well known and conventiona~
tests and criteria, e.g. reduction in shrink temperature, T.; susceptibility to enzyme attack, e.g. collagenase;
extractability of degradation products, e.g. collagen fragments; and decrease in physical properties such as tensile strength.
In accordance with an embodiment of the present invention, the biological tissue may be crosslinked prior to irradiation. Any crosslinking reagent may be use~, preferably a reagent which stabili2es the tissue against subsequent in YiVo enzymatic degradation, typically by crosslinking collagen in and on the biological tissue.
Suitable crosslinking reagents include, but are not limited to glyoxal, formaldehyde, and glutaraldehyde.
The preferred crosslinking agent is glutaraldehyde.
The crosslinking can be carried out in any desired method. Many such methods are described in the prior art. Generally, the crosslinking step comprises immersing the tissue in a reagent solution for from a few ~inutes to several days depending upon the desired degree of crosslinking. The solu~ion may include one or a number of crosslinking reagents, such as, for example, glutaraldehyde, formaldehyde, glyoxal, and/or dialdehyde starch. The rate of crosslinking reaction can be controlled by controlling the concentration of 30 crosslinking reagent and, to a lesser extent, by controlling the pH and/or the te~perature of the crosslinking reagent. For example, the concentration of glutaraldehyde may be from about O.OOl~ to 8.0~ volume to volume (v/v), preferably less than about O.l-~ v/v glutaraldehyde.
The solution is typically buffered with any suitable buffer. suitable buffers for use in the practice of the f, ' 2 1 69478 WO95123616 PCT~S9~/0283 invention are those buffers which have a buffering capacity sufficient to maintain a physiologically acceptable pH, e.g., a pH between about 6 and about 8, and do not cause any deleterious effec~s to the biomaterial or interfere with the treatment process.
Exemplary bu~fers include, but are not limited to phosphate-buffered saline (PBS), and organic buffers, such as N-(2-hydroxyethyl)piperazine-N'~ ethanesulfonic acid) tH~PES~ or morpholine propanesulphonic acid (MOPS);
and buffers which include borate, bicarbonate, carbonate, cacodylate, or citrate. In a preferred embodiment the solution is non-phosphate buffered, more preferably, citrate buffered at pH 6.4 or HEPES buffered at pH 7 . 4 .
Time and concentration are, of course, related and considerable variation in both are well known in the art.
In a typical protocol according to the in~ention, the biological tissue may be exposed to the fixing solution for a time and at a temperature sufficient to induce crosslinking of the collagen in and on the biological tissue. Por example, the biological tissue may be exposed to a buffered glutaraldehyde solution from about 4C to about 37C, preferably at about 20C; at a pH fro~ about 6 to about 8, preferably 6.3 to 6.5; and for a period up to about l0 days, preferably from about 2 to about 5 days.
In accordance with the invention, one skilled in the art ~ill recognize that certain parameters in the treatment protocol may be varied according to achieve a particular purpose. These parameters include, but are not limited to glutaraldehyde concentration and solution composition, pH and ionic streng~h, time and temperature of biological tissue exposure to glutaraldehyde, the ratio of tissue to volume of solution, and the biological tissue configuration during the initial fixation.
An embodiment of the invention may include exposing the crosslinked biomaterial to one or more bioburden reduction agents, typically for up to about l0 hours, ` 2 1 69478 wO9Sn~616 rC~S9~0283 preferably for about 2 to about 4 hours. For example, a porcine heart valve treated with glutaraldehyde as noted above may then be exposed to a buffered solu~ion containing 1-5% glutaraldehyde, 1-6~ formaldehyde, and 15-25~ ethanol. Typical buffers include PBS, HEPES, and citrate buffers.
In accordance with an em~odiment of the invention, the biomaterial, treated with glutaraldehyde as noted above, may then be exposed to one o~ more ~eagents designed to reduce or inhibi~ calcification of the biomaterial after implantation. For example, the crosslinked biomaterial may be exposed to an alco~ol and/or an aluminum salt in order to reduce or inhibit calcification. In an exemplary process, the crosslinked biomaterial may be i~mersed in a solution containing greater than about 50~ of a lower aliphatic alcohol such as ethanol for a period sufficient to ~ender the biomaterial resistant to calcification, typically up to about 96 hours.
Typically, the crosslinked biological tissue is then rinsed, using, for example, any suitable ~insing or laving ~aterial. In a preferred embodiment, the rinsing agent is sterile, physiological saline.
The tissue may be rinsed with many volumes of sterile, physiological saline over a period of approximately 24 hours, or until the concentration of residual processing che~icals in the tissue are below levels which are considered to be toxic (approxi~ately l ppm).

The biological tissue may then be placed or packaged in a container. In accordance with a preferred embodiment of the invention, the biological tissue is packaged and sealed, in physiological saline, in its final container prior to terminal sterilization.
Packaqing preferably means placing in a container suitable for storage and/or shipping.

21 6947~

wossn3616 PCT~S9~10283 1~
The container may be constructed of glass or polymeric plastic Suitable plastic materials include polye~hylene; acrylates such as polymethyl methacrylate and polymethyl acrylate; polymethyl pentene-l; polyvinyl chloride; vinyl chloride-vinylidene chloride copolymers;
polypropylene; urea-formaldehyde copolymer; melamine-formaldehyde copolymer; polystyrene; polyamide;
polytetra~luoroethylene; polyfluoro~richloroethylene;
polycarbonates; polyesters; phenol-formaldehyde resins;
polyvinyl butyryl, cellulose acetate; cellulose acetate propionate; ethyl cellulose; polyoxymethylene; and polyacrylonitrile. In a preferred embodiment, the container is constructed of polypropylene, polyethylene, and/or epoxies. lt is intended that the in~ention should not be limited by the type of container and seal being employed; other materials may be used, as well as mixtures, blends, andlor copolymers of any of the above.
The crosslinked, packaged biological tissue may then be sterilized, as noted above, or it may be stored ~or up ~o about a year or more prior to sterilization.
In accordance with the invention, storage includes long term storage, e.g., six months, twelve months, or for up to five years or more.
Some conventional techniques use glutaraldehyde as a sterilization agent in the packaged product sent to the surgeon. Such sterilization agents must be rinsed from the tissue prior to implantation. However, some of the embodiments according to the in~ention provide a product that requires no rinse prior to implantation. Residual 30l chemicals, such as glu~araldehyde, used in the pre-packaging processing of the biological tissue are ~emoved from the product prior to packaging, and the packaged tissue is terminally sterilized.
The present invention also includes a biological tissue vhich has been sterilized using E-beam radiation and has improved hemodynamic properties. In a preferred embodiment of the invention, the biological tissue is `` 2169478 WosS/236t6 PCT~S9~/0283 crosslinked with a suitable crosslinking reagent and irradiated with a bea~ of accelerated electrons to sterilize the tissue. As noted above, the tissue may be terminally sterilized after it has been sealed in a sterile container.
In accordance with the invention, tissues which have been exposed to E-beam radiation may be softer or more pliable, may exhibit a greater rànge of movement fo~ some of its movable parts, e.g., the leaflets of a heart valve; and increases the bio~ogical tissue's long-term durability.

ExamPle~

ExamDle 1. Glutaraldehyde pre-treatment. Fresh tissue (e.g., blood vessels, hearts, heart valves, or pericardium) are procured from a local processing facility (bovine, porcine, ovine, etc.) and received in physiological saline (0.9% sodium chloride) on ice. The tissue is either dissected immediately or placed in fresh sterile saline and refrigerated overnig~t. Ex~raneous tissue such as adipose, skeletal muscle, myocardium, bone, trac~ea, etc., is carefully removed from the tissue of interest. The tissue is then again washed and immersed in fresh sterile saline.
Although this technology works to varying degrees at a range of glutaraldehyde c~ncentrations, approximately 0.03% provides radioprotective properties and the crosslinking time fits reasonably well within a manufac~uring schedule. For lo.0 liters of 50 mM citrate buffered 0.03~ (v/v) glutaraldehyde;

step 1) A 50 ~M citrate buffer solution is prepared per the following formula (10 liters):
To 9.0 liters of sterile, de-ionized water, add:

`,_ 216q478 WO gS123616 140.0 gra~s of Sodium Citrate 5.0 grams of Citric Acid Monobasic 38.6 grams of Sodium Chloride Bring the vol~me of the solution up to 10.0 liters with sterile, de-ionized water Step 2) To 9.O liters of the 50m~ citrate buffer solution prepared in Step 1, add 6.0 lo milliliters of 50~ Biological Grade Glutaraldehyde Bring the solution volume up to 10.0 liters using the 50mM citrate buffer solution prepared in Step 1.
Step 3) Adjust the p~ of the solution to 6.40 + 0.05 using hydrochloric acid or sodiu~ hydroxide.

The tissue is then immersed in the glutaraldehyde solution, at room temperature (20-25C) for the crosslinking reaction. As fixation time progresses, the number of crosslinks increases, as shown in the form of a shrink temperature curve (See Figure 5). The concentration of glutaraldehyde in solution decreases as it is consumed by the tissue in the form of poly-glutaraldehyde crosslinks. See Figure 6. Therefore, it may be necessary to replenish the fixation solution at intervals throughout the crosslinking reaction. Because a major~ty of the crosslinks are formed early, it is recommended to change the solution approximately eight hours following the onset of the reaction, then daily thereafter.
T~e exposure of tissue to the glutaraldehyde solution proceeds for a period of time ranging from 24 to 120 hours, depending on the concentration of glutaralde~yde in the solution. In general, a high ` _ 2 1 69478 WO95123616 ~CT~S~/0283 glutaraldehyde concentration corresponds to a short fix~tion ti~e; a low glutaraldehyde concentration corresponds to a long fixa~ion time. For a 0.03~
solution, an exposure time of approximately 72 hours is necessary to maximize the crosslink density within the interstices of the ti5sue. This corresponds to a shrink temperature of approximately 80-89C, depending on the type of tissue used.
When the crosslinking reaction has ended, the tissue lo is submersed in a solution containing 2% (~/v) glutaraldehyde, 3~ (~Iv) formaldehyde, and 20% (v/v) ethyl alcohol. This multi-component sterilant reduces any residual bioburden on the tissue prior to rinsing ~nd packaging.
The tissue is then thoroughly rinsed with sufficient sterile saline to remove all processing chemicals. This typically requires applying four or five 10 liter aliquo~s over a 24-hour period. ~he exposure time must ~e watched carefully, since diffusion of residuals from the tissue is a time-dependent phenomenon. After the final rinse, the tissue is placed in a sterile container (valve jar, vascular graft ~ial, etc.) and fille~ with sterile saline. The package is then permanently sealed.
~ote: all manipulations of the tissue subsequent to the bioburden reduction process with the multi-component sterilant should be performed as aseptically as possible to minimize the extent of contamination prior to E-~eam sterilization.

Example 2. E-beam radiation. Porcine aortic leaflets were crosslinked with 0.01%, 0.1%, or 0.6%
glutaraldehyde using the protocol described in Example 1.
The non-control leaflets were then exposed to 25 kGy E-beam radiation. Table 1 is a summary of the data from an experiment designed to demonstrate how collagen integrity is preserved by E-beam irradiation of tis ues cross}inked in low-concentration glutaralde~yde. As shown in Table wos~n3616 PcT~sss/028 1, a ~eduction in c~rink temperature was shown for tissues crosslinked in low-concentration glutaraldehyde and sterilized by exposure ~o E-beam radiation.

T~ble 1.
Shrinl~ T~ r ~ e (-C): Glutar~3~- G~ ` '-~ Porane Aortic Leaflets nd Po~ '^d -" (25 kG~) 0.01% C;1-'J ' ~de ¦ 0.1% rl" '' ' ~rdl~ 0.6X fil~ e Sample Control E-~eam Cont~l E-Beam Control E-Beun 1 87 82 86 81 8? 82 2 86 82 87 ~1 88 82

3 86 81 86 81 87 82

4 86 82 87 82 88 82 ~ 8? 81 88 82 6 ~ 86 81 88 82 . Eii ~ E 9~ ~ i iii~ ~ ~Re ~ ~ l l ~1 9~1 ~ ~ ~ E ~ ~ ~ ~ 86 81 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
a~a ~s ~ ~3 ~ ~ 39~ ~ ~ ~ ~ 9 ! ! ~ ~1 ~12 8 ~ ~ iE ~ ~ ~ 86 81 ~ ~ 3~ ~ ~3 ~ a~ ~ z ~ ~ I @ ~ ~ ~ E~
9 ~ !~! ~ 1~ ~11 ~ ~ ~ 87 82 1~ 3!~ ~ ~ _~ 82 ~ ~ 1~ ~3 11 ~ ~1 ~- 81 ~ ~ ~ ~ ~3 Mo~ 86 82 86 81 88 82 S~ 0.5 0.5 0.5 0.5 o.~ o 4 ~ S ~ 6 Example 3. For 10 MeY electrons, the ratio of ~axi~um to ~inimum dose is typically a~out 1.3:1. This phenomenon is illustrated in ~igure 2 which shows the distribution of dose with depth when a material is irradiated wi~h electrons at different energy levels.
For 10 MeV electrons in unit specific gravi~y material, the maximum dose is achieved at a depth of about 2.3 cm. The dose is a~ou~ the same as the dose at the surface at a depth of 3 cm, and is practically zero at a depth of 5 cm. For 10 MeV electrons the maxim~m 2169~78 wo9s/23616 PCT~Sg~M83 dose is abo~t l.33 of the dose at the entrance and exit surfaces, or a ratio of about l.3:l.
The same entrance and exit dose is achie~ed for materials with an areal (unit density) of about 3.0 g/cmZ
for slngle-sided irradiation with lO MeV electrons.
However, the primary beam causes additional charged particle fluence of electrons in the material. The result is a buildup of dose within the material, particularly in the center. For example, if the sur~ace dose is about lO kGy and the exit dose is about lO kGy, the build-up in the center may be about 13 kGy. This contrasts sharply with gamma irradiation, in which the gamma rays transfer energy by Compton scattering collisions with atomic electrons. Here the probability for compton scattering is lo~, allowing the gamma rays to penetrate relatively long distances in materials before scattering. Therefo~e, gamma rays deposit energy in materials over large volumes so that penetration is high, but dose rates are low (typical~y about ~0 kGy/hr maximum, 4 kGy/hr average; 50 cm depth in unit-density material).

ExamPle 4. An experiment ~as perfo~med to calculate the maximum dose experienced inside a biological vascular graft package due to build-up~ Bovine carotid arteries were cleaned and crosslinked with 50~ citrate-buffered 2~ glutaraldehyde. The grafts we~e irradiated, immersed in saline inside their glass tubes, with 9.2 + O.Ol MeV
electrons from the I-lO/l Linac at the Whiteshell Laboratories. The tubes were laid on their sides and exposed to the scanned beam fro~ above. Several dose studies with radiochromic dye dosimeters (Far West Technology) placed above and below the tubes sho~ed that the dose on top of and immediately underneath the tubes was the same to within a few percent. This indicates that the glass tubes and their contents are approximately the optimum thickness for single-sided irradiation with ` 2169478 WO gS~23616 PC~lUS9ilo2~3 electrons of this energy (-~g-cm'2). This means that the dose at ~ome points within the tubes could be as much as 33~ higher.
A sheet of red polymethylmethacrylate (red PMM~ or red acrylic) was placed under one set of three tubes, which were wrapped in plastic bubble wrap to prevent mechanical da~age. The tubes and PMMA were irradiated in the sa~e way as before with 9.2 MeV electrons.
The PMMA darkened in proportion to the dose it recei~ed. Therefore, if the calibration curve relating the absorption to dose is known the dose dist~ibution in the plane of the red acrylic sheet may be determined.
Normally, only the rela~ive dose distribution is of interest.
Absorption and scattering of electrons by the overlaying tubes was plainly visible in the PMMA
following irradiation. A co~puter cont~olled travelling densitometer (Therescan, Theratronics Limited, Xanata, ontario) was used, first to scan across t~e PMMA and then to plot isodose cur~es over the surface of the PMMA
sheet.
~igure 3 shows densitometer traces across the PM~A
sheet perpendicu~ar to ~he tubes. These transect the PMMA sheet at five different points along its length (i~e., zt -20, -10, 0, lO and 20 cm). The relative light absorbance, which is proportion~l to dose, is shown on the legend to the left in Figure 3. This indicated a maximum to mini~um dose ration of 1.o to 0.6~, or about 1.5.
Isodose contours for the same PMMA sheet are plotted in Figure 4. Each of the five transects in ~igure 3 were normali~ed to maximum absorbance for that transect. The isodose contours are normalized to the ~aximum absorbance at any point on the sheet. The lowest d~se contour ~S surrounds a small area in the lower left of the figure.
T~is contour received 65~ of the maximum does, so t~e max/~in ration is 1.54.

woss/236l6 PCT~S9~tO2835 The results of the study with PMMA do not contradict the study with the Far West dosimeters, which indicated that the dose im~ediately above and below the tubes is about egual. In fact, PMMA results indicate that, except for a few cases, the dose registered in the PMMA between the tubes is approximately the maximum 133~ of the dose underneath the tubes due to buildup in the packing between the tubes.
The exceptional case may be important. The 65%
maxinum dose contour surrounds an area of the PMMA which was underneath the cap of the tube ln this case, the glass of the tube is thicker to accommodate the screw tap and the cap itself adds to the a~ount of material the elec~rons must penetrate. Mo~eover, at these points the scanned beam is directed at a slight angle from the perpendicular to the axis of the tube and must penetrate slightly more material in the slant-wise direction. This does not necessarily mean that the inside of the tube is not getting sufficient radiation to result in sterilization.
In summary, the grafts, as presently packaged and irradiated in glass tubes, are receiving approximately equal dose on the upper and lower surfaces of the tubes and about 133% of the dose on the upper sur~ace inside the tubes at the loca~ion of the grafts. The exception to this is that the dose under the caps appear to be approximately 65~ of the maxi~um does received by the grafts, or about 86% of the dose at the upper su~face of the tubes. If the top surface were to ~eceive 25 kGy, the dose to the grafts could be as high as 33 . 3 ~Gy and the dose under the cap end of the tubes would be about 21 kGy.

ExamPle 5. Experiments have shown that glutaraldehyde-crosslinked tissue, exposed to E-beam radiation, exhibits enhanced hemodynamic performance characteristics, such as flexibility. Evidence of `` 2169478 WO95/2361G PCTIUS9~/02835 increased flexibility is provided by measuring pressure drop across the hea~t valve (the change in pressure from the inflow side of the valve to the outflow side), as sho~n in Figure 8. Enhanced flexi~ility is also shown by measuring the effective orifice area, the cross sectional area through which blood flows, as shown in Figure 9.
These tests show that exposing ~eart valves to E-beam radi~tion results in softer leaflets w~ich tend to open ~ore readily and to a greater extent than non-irradiated valves. This provides both short-term and long-term benefits to the recipient because a largeF effective orifice area results in greater cardiac output and ~herefore, an increase in efficiency of cardiac activity and a decreased tendency to develop cuspal fractures leading to eventual calcification and valve failure.
Eight heart ~alves were glutaraldehyde crosslinked and exposed to E-beam radiation as shown in Examples 1 and i. The pressure drop acrosc the heart valve before subjecting the heart valve to E-beam radiation ~as ~omp~red to the pressure drop after subjecting the heart valve to E-beam radiation. Figure 8 graphically illus~rates that the pressure drop decreases when tested on a steady state in vitr~ flow ~ester. As a reference point, the pressure drop for a straight, unobstructed tube would be zero.
Figure 9 compares t~e effective orifice area before and after exposing the heart valve with E-beam radiation, and shows that the effective orifice area increases following E-beam ~adiation.
Effective Orifice Area determinations were made by placing test valves in a Pulse Duplicator system. The Pulse Duplicator is capable of calculating a number of valve-related functions by measuring pressures and flow rates at strategic locations within a simulated heart containing the test valve.
Effective Orifice Area (EOA) is defined as follows:

.

WosSI236~G PCT~S9~10283 EOA=Q~,/(51.6 ~P), expressed in cm2, ~here Q~- root mean square flow rate obtained during the period of positive pressure drop, in ml/second ~P= mean positive pressure drop, in mm Hg The theory behind enhanced hemodyna~ics in irradiated tissue heart valves involves the di6ruption of molecular bonds which hold t~e collagen triple ~elix intact. The intramolecular crosslinks offered by this technology serve as reen~orcement to the collagen backbone as its o~n structural frame work is weakened by the radiation. A dose of 25 kGy, in the presence of sufficient intramolecular crosslinks, wea~ens the protein frame~ork to sufficiently render the tissue ~ore flexible, yet the tissue performance improves.
Similar results have been obtained every ti~e these two experiments ~ere repeated. While the exact mechanism is unknown, it is theorized that a scission reaction occurs within the collagen molecule. Bonds that hold the collagen chain together appear to be broken when subjectin~ a tissue to E-beam radiation. However, the presence of intramolecular glutaraldehyde crosslinks appears to keep the primary structure of the collagen molecule intact, thus maintaining the integrity of the softened tissue.

Exam~le 6. The major criticism of radiation as a sterilization method for biological tissues is its effect on long-term durability of the product. The FDA
currently requires that tissue valves demonstrate the ability ~o withstand 200 million cardiac cycles on an accelerated wear tester. This translates to approxi~ately five years of real time. At some point in the ~uture, 380 million cycles of the same testing may be required.

-` 2 1 69478 _ W09~36t6 ; PCT.~S9~10~3 We performed an eXperiment to determine the ef~ects of E-beam radiation on the wear-resistance of tissue Yalves. ~our groups of valves were tested:

S Group 1 CrosslinXed in 0:.03~ glutaraldèhyde;
stored in 0.5~ glutaraldehyde lE-beam negative control~.
Gro~p 2 Crosslinked in 0.03~ glutaraldehyde;
rinsed for removal of residuals;
stored in 0.9% sodium chloride;
E-beam sterilized, 25 kGy.
Group 3 Crosslinked in 0.03% glutaraldehyde;
treated with anticalcification process;
rinse~ for removal of residuals;
stored in 0.9~ sodium chloride;
E-~eam sterilized, 25kGy.
Group 4 Crosslinked in 0.5~ gl~taraldehyde;
rinsed for remo~al of residuals;
stored in 0.9% sodium chloride;
E-~eam sterilized, 25 kGy (conce~tration negative control).

Results of this experimen~ are located in Table 2 belo~. These results clearly indicate that, co~pared ~o control valves (Groups 1 and 4), exposing the tissue valves to E-beam radiation does not have a negative effect on durability after in Yitro testing at 389 million cardiac cycles. The group with the best wear data, in fact, was the group that had been exposed to E-beam after a treatment fcr anticalcification.

W095/23616 PCT~S9~tO28~5 T bc2.
~kD~ G~nC~d~ ~ Wo~Te~g:
~B~m.No ~BbYn ~r~~ D'Nwm~of ~ ~O~ Ar~ -a'--Yal-es G~upl 4 61~gcbol~(~ 1~m) 2 sm~ll holes (~ Imm) 2 l~rgc ~rs ~~ nctS (2-6 mm) I ~11 ~brasion I ~lvc with no obscr.~ r Gmup 2 4 2 l~r~e holes S small boles I ~ br~sioD
I v~lvc wi~h no observed vvear G~up3 6 3sm~l hol~
4 v~lYes with DO obsc~cd wear G~up~ 3 3hol~(0.5to3mm) 2 v~lvcs wi~h ~o o'oserved we~r ExamDle 7. To determine if there is a significant difference in the response of Bacillus pumilus to equivalent doses of gamma and E-beam radiation, a population of B. pumilus ~as irradiated in liquid s~spension wi~h gamma and E-beam radiation, then the surviving fraction of the population at a series of doses was deter~ined. At a dose of 6 kGy, there was approximately 100 times more surviving organis~s that were gamma irradiated than were E-beam irradiated ( surviving fractions of 10~ vs . 10, respectively).

Example 8. There has always been some concern as to the effects of E-beam radiation on the micro-structure 25 of tissue. The issue of preservation of "collagen crimp~l, or the natural ~aviness of collagen is very important in providing superior performance and durability in any bioprosthetic val~e. An experiment was performed to examine the effects of dynamic, or pulsatile s 2 1 69478 ~09Sn36l6 ~ PCT~S9510283 fixation (with and without E-beam), on the morphology of porcine aortic valve leaflets.
Three groups of tissue were prepared for this experi~ent. one group contained tissue crosslinXed with 0.03~ glutaraldehyde in a pulsatile fashion, rinsed of all residuals, and sterilized wit~ 25 kGy electrons. A
second group was treated the same as the fir~t group, but was not steri}ized ~it~ E-beam radiation. The control group contained leaflets that most closely represented "natural" valve leaflets: crossl~nXed under "zero-pressure" conditions ~o maintain integrity of all cellular and acellular components.
Each group of leaflets, for~arded to an independent agency for evaluation, were found to have virtually indistinguishable morphology, and that there was no consistent effect of either dynamic fixa~ion or of a sterilizing dose of ionizing ~adiation on the structure of the valves. Furt~ermore, there were no consistent differences a~ong the valves in any of the following:
collagen crimp, collagen crispness, internal valve spaces, amorphous extracellular matrix, or cellular autolytic features.

Exam~le 9. The increase in temperature during the E-beam process was ~easured. Thermocouple leads were inserted through small holes drilled in the caps of two packages containing a biological vascular graft packaged in saline. The leads were then positioned between the graft tissue and the polycarbonate mandrel to measure the te~perat~re at ~he mandrel/tissue interface dur~ng E-beam exposure. The results of the experiment indicate that the temperature rise was approximately 7C over ambient temperature, resulting in a final temperature of approximately 27C.
Examples lo and 11. T~e effects of glùtaraldehyde fixation on bovine vascular tissue and possible , wo9sn36l6 ~ PCT~S95/0283 destabilizatio~ by ionizing radiation can be evaluated be determining the denaturation temperature of the substrate. A convenient method of determining this value is by measuring the shrinX temperature (T~) of the tissue, w~ich increases with an increasing number of crosslinks.
Glutaraldehyde crosslinked vascular tissue, follo~ing exposure ~o ionizing radiation, has demonstrated a loss in thermal stability. In previous gtudies, a decrease in T, of approximately 6DC had been noted following a 2.5 Mrad dose of electron beam (~-beam) irradiation. Several modifications to the storage solution including the use of radioprotectant compounds sodium thioglycolate and mercaptoethylamine ~M~A), catalase (a hydrogen peroxide scavenger), and alternative buffers, have in some cases minimized t~e T, depression after 2.5 Mrad of radiation exposure. ~he objective of these studies was ~o test and identify one or more methods of packaging and radiation sterili2ing biological tissue while keeping tissue damage to a minimum.
Example lo. Twen~y median artery grafts were ficin digested and glutaraldehyde crosslinked as follows:
the grafts were crosslinked with 0.01% glutaraldehyde for 112 hours, and then pre-sterilized for five hours in 2 glutaraldehyde. The grafts were then aseptically packaged in sterile 0.9~ sodium chloride and allowed to remain on the shelf for a period of 9 days for diffusion of residual glutaraldehyde from the tissue. Grafts were then placed into a sterile tank containing 16 liters of sterile saline for fu~ther rinsing of glutaraldehyde residuals. Fol~oWing a 3-~our rinse in sterile saline, the grafts were packa~ed for E-beam sterilization. ~ach graft was placed in a polye~hylene pouch and filled with a 50mM citrate-buffered saline solution at pH 6.4 and radioprotectant additives as follows:

5 packages - O.OlM sodium thioglycolate WO95/~616 PCT~S9~10~83 S packages - O.OlM ~EA
5 packages - O.lM MEA
5 packages - control (citrate-~uffered saline only) The 15 non-control packages were then exposed to 2.5 Mrad E-beam irradiation.
Traditionally, radioprotectants ha~e been ad~inistered to animals or culture media immediate~y prior to irradiation to mini~ize its effects. Most of the compounds used in early radioprotection contained either -SH or -NH2 groups because of their ability to absorb energy e~itted by radia~ion sources. The exact mechanism of pro~ection, however, is still un~no~n. In the early 1950s, approximately three thousand compounds were tes~ed for effectiveness as radioprotectants and for toxicity. Of those tested, ~-mercaptoethylamine (MEA or cystea~ine) ~as found to ~e the most effective as an in situ radioprotectant used with tu~or radiotherapy. T~e compound bas been administered intravenously to humans in doses of up to 500 mg, twice per day, for thirty days with no ill effects.
Another thiol compound, ~odium thioglycolate, has been used as a radioprotectant ~ith the ~amma sterilization of culture media to eliminate the need for aseptic filling. Sodium thioglycolate has been used at a level of o.OlM. It was preferred over other agents because it is nontoxic and does not significantly reduce the efficiency of the sterilization.
As the T, results displayed in Table 3 suggest, there was not ~uch protection afforded by either of the radioprotective agents. The mean T, value obtained for the 0.lM MEA (75.6C) was, in fact, lowe~ than the samples with O.OlM MEA (77.~C).

`` 2169478

6 PCTtUS9~0283 T~ble 3.

T~ble 3. S~ k T~ Resultc - E~a~ple 10 ~_ T (C) aftcr 2 5 M~at E-Be~
.
Sample Controls No 0.01~ 0.01 0.1 Number (DO E-beam) PYC ~ ioglycol~.te ~ M
l) MEA 1~EA
IA 8i.6 77.2 78.4 7?.2 7S.2 IB 81 2_1 77 6 7~.6 75 6 10 2A 83.677.6 ?8.0 77.2 77.2 2B 83.2_¦ ?8.8 ~7.2 76.8 3A 82 877 2 76.4 7?.2 76.4 3B 83.2 _ 77.2 78.0 75.2 4A 82.87~.2 78.0 78.4 75.2 15 4B 83.2~_~ 79.6 ~8.0 74.8 ~;A 82.817.6 76 8 ~7.2 ~4.8 SB 82.4_I 77.6 76 8 ~5.2 6 ~ 77.6 Mean 82.7'7~.4 77.8 77.5 75.6 2 0Std. 0.8 0.2 0.9 0.~ 0.9 D~. .
~ After NA~ 5.3 4 9 5.2 7.1 E-be~un Imean) 2 5 *Nol Applicable Example 11. In this example a second batch of bovine ~edian arteries, processed as in Example 10, ~as used for E-beam testing. The sodium thioglycolate concentration was increased from the previous batch from 0.01 to O.lM. Catalase was also added to two of the test groups to decompose hydrogen peroxide (H202), a ~y-product of E-beam irradiation which may be deleterious to graft wall integrity. The concen~ration f ~2 generated by Wog~l236l6 PCT~Sg~10283 ionizing radiation in polyethylene containers should theoretically be approximately 50 x 10~ ~oles per liter~
The following informa.tion was pro~ided with the lot of catalase used in this study tSigma, lot lOOH382g, derived from Aspe~gil~us niger):

24 mg protein/ml stock solution 7080 units enzyme/mg protein 1 unit catalase will decom~ose 1.0 ~mole H22 per minute at pH 7.0 The buffer used in the storage solution was changed in this example ~rom citrate to HEPES, tpH range of 7.2-7.4). HEPES is a commonly-used biological buffer used to a~hieve this particular pH range. Based on T, results obtained with MEA in Example 10, it uas not used in this phase of the study. Rather, the concentration of sodium thioglycolate was increased ten-fold to O.lM.
Damage occurring in radiation-sterilized culture media has been attributed to the formation or accumul~tion of peroxides. The damage to the collagen in or on this product, indicated by a decrease in T" may be caused by the same mechanism. The addition of the radiation-resistant enzyme catalase, which is a peroxide scavenger, has been shown to reduce ~2 ' As in Exa~ple 10, the use of thioglycolate, with and without the addition of catalase, provided minimal protection based on T, (means of 76.8c and 77.4C
respectively). The group containing HEPES buffer, however, resulted in a mean T, value of 78.5OC, which is only 2.8 degrees lower than the control (no E-beam) value of 81~3C. The data gathered in this phase of the study suggests that damage caused to the tissue could be minimized more effectively by buffering in the proper pH
range than by using traditional radioprotective agents.
The following calculations were then applied to determine the amount of catalase necessary to deco~pose Wo95123616 ~CT~S~10283 the ~2 theoretically generated by the irradiation process (4.5 liters of packaging solution was needed for the batch):

Total amount of H202 generated in 4.5L:
4.SL x (50 x 104moles/L) = 2.25 x 101 moles Units of catalase to decompose ~2 in 4.SL packaging solution:
2.25 x 104 moles (1 ~mole~10~ mole)(l uni~
catalase/l ~mole) = 225 units Units of catalase per ~1 of stock solution:
7080 unitslmg x 24mg/ml stock solution = 169,920 lS units/ml stock solution Volume of stock solution necessary for 4.5L packaging solution:
225 units (1 ~1 stock solution/169,920 units) =
1 . 3 X lo-3 ml or approximately 2 ~L stock catalase solution per 4.5L packaging solution Units of catalase present in each graft pacXage:
(2 ~L stock solution/4.5L packaging solutio~)(1 ml/1000 ~L)(24 ~g protein/ml) tO.15L packaging solution/package)(7080 units catalase/mg protein) - 11 units catalase/pac~age (volu~e of catalase per package was rounded to 2 ~L due to the'limits of the measuring device) The effectiveness of the radioprotectants was e~aluated by T, testing. ~our combinations of storage solutions were prepared as identified in Table 4 below:

` 21 69478 WO gSfZ36lG PCI/US9~/0283 ~ble 4.

Table ~. Test Groups F ~!ie 11 Group H~p<s ¦ T~ y~~'~ e Cahlase ¦ E-Beam 1 (A~) X _~ X
2 (~-N) X X ~ ~ ~ X
3 (O-U) X X X X
4(V-~B) X X X

Results of the T~ analyses are displayed in Table 5 below:

T~bkS.Su~Tffnp~u~ Rk~

Group Number T,(-C) ~ trol 1 ~EIEPES, E-bc,am, 2.5 78.5 + 0.6 D='7 2.8 M~d) 2 ~IEPES, Thi~ , 77.4 + 0.8 n=7 3.9 E-~e~m, 2.5 M~t) 3 (HEPS, 1~ ' , 16.8 + 0.9 n=7 4.5 l~e, E-~m, 2.5 Mrad) 4 (HEPES, 1~jG~ 81.3 + 0.8 n=7 N/A~
C~t.alase, ~o E~
c~nlrol group 2 5 ~Not Arpllç~le Examples 12 and 13. Examples 12 and 13 involve the use of bovine pericardial tissue in minimizing the destructive effects of the radiation. Pericardium was used as a substitute for vascular tissue for these examples for the following reasons: much less preparation time is necessary and therefore, more samples may be prepared per batch, the tissue possesses a very high collagen content (approximately 90~ versus 45% in ~he carotid and median arteries) ~hich assures accurate and consiste~t results, and the results should be easily translated to vascular applications.

wossn36l6 PCTlUS9~/0283i ExamPle 12. In this Example, the tissue was e~aluated after storage in various biological buffers without ~he addition of the radioprotecti~e compounds noted in Example 11.
Four fresh bovine pericardial sacs were received in physiological saline (0.9~ sodium c~loride) on ice.
The tissue was placed in fresh ~terile saline and refrigerated overnight. Adipose tissue was carefully removed from the epicardial surfaces and they were again washed in sterile saline. One hundred thirty-three 2cm x scm sections, which represents the normal T, graft test sample size, were cut from the pericardial tissue. The samples were evenly divided between two la~ge beake~s, each containing 3 liters of 50mM citrate-buffered 0.05 glutaraldehyde. The tissue samples were allowed to crosslink in the glutaraldehyde solution for approximately 90 hours. They were then subjected to a four-hour 2~ glutaraldehyde bath for sterilization. The tissue samples were then divided into ten test and control groups packaged in lSOml of the following solutions, shown in Table 6. All packages containing HEPES or Tris were adjusted to pH 7.4. Each group was prepared in duplica~e, one for E-beam and one for control.
Tib~6.~

Group 0.9% Sodium 11.0 u~its 0.2M HEPES 0.05~ Tns C:hloridc C~t.alase X ~ ~ ~ ~ ~ ~
. Z~ b~ =~ ~a E~
2 ~ ~ ~ ~ X
~ ~ ~ ~ ~ _ 30 3 1~ ~ X X
4 _ X ' X
S ~ X

`` 2 1 69478 wo95n36l6 PCT~S9~/0~83 Each group contained approximately 13 sa~ples of-crosslin~ed pericardium. samples were placed into radiation-resistant polyethylene pouches, filled with the appropriate solution, and heat sealed. Each pouch was then placed into a secondary pouch to assure against leakage. The tes~ samples were E-beam i~radiated at 2.5 Mrad and tested for T,.
The use of boYine pericardium for this example allowed a much greater sample size for each test and control group. The results displayed in Table 7 suggest that the mean T, depression observed for tissue samples s~ored in 0.05M Tris buffer was less than any method of radioprotection attempted to date ~ = 2.5C); the ~ean T, value for these samples was 80.2C. The experiment described in Example 13 was then designed to further evaluate the effects of Tris when used as a packaging solution for E-beam and gamma irradiated products.

` 2169478 WO 95/23616 PCTt~S95tO283 Resul~s of the T, analyses:

Table ~. Shrink T~.pc~l~ c ~ults - F ~ ~ 'e 12 Group ~umkrMam T ~ ~ ' d De-i~tion in T, due to (-C) E-beam ('C3 IA (sal~e, co~tn~l) 82.9 O.g lB (s~ e, E~ m, 78.7 0.5 4.2 2~1~.I~) 2~ ~HBES, 83.3 0.7 1 0 C~D~
2B (HEPES, E- 78.7 0.7 4.6 ~e~m, 2.5 ~JYI) 3A ~EIEP~S ~ 83.0 1.0 catalase. control) 3B (HEPES ~ 18.8 0.6 4.2 lase, E-~m, 2.5 Mr~d) 4A (sali~e + 82.9 0.5 4B (u~r~e + 79.2 0.7 3.7 cat~l~c, E-b~m.
2.5 Mr~) 5~ (tris, cootrol) 82.7 0.6 SB (tris, E-t~e~m, 80.2 1.03 2.5 2 5 2.5 M~ad) Example 13. Pericardial tissue was received and crosslinked as described in Example 12 above ~ased on T, results received from the tissue ~-beam sterilized in ~xa~ple 12, only tvo of the solutions were prepared for this example: 0.9~ sodium chloride and 0.05M Tris.
Approximately 30 samples of tissue were packaged in each of the two solutions in p~lyethyle~e p~uches and gamma sterili2ed at a dose ranging from 3.1 to 3.4 Hrad. A
duplicate set o~ samples was E-beam irradiated under a lo ~e~ accelerator. The 10 MeV accelerator is capable of pe~etrating and sterilizing tissue packaged in a standard glass vi~l or a vial similar dimensions. The samples were E-beam irradiated at a dose ranging from 1.09 to `` 2169478 . .

wo95n36l6 . PC~S9510283 1.42 Mrad. T~e samples were then evaluated for T,. ~ive bo~ine carotid artery gr~fts were ~lso subjected to each process to determine if the addition of Tris to the packaging solution ef~ects tissue damage detectable ~y T,.
The data presented in Tables 9 and 10 below suggests that t~ere is less of a T, depression when Tris, rather than ~aline, is used as the storage solution for carotid artery grafts with gamma irradiation.
There was not a significant difference in T, values obtained using ga~ma irradiated pericardium packaged in saline or Tris. The reason for the ~inimal depression observed for tissue packaged in saline cannot be explained, especially since graft tissue irradiated simultaneously in ~he same box exhibited a mean depression of 4.5~c. The actual dose administered to this batch of tissue ranged from 3.1 - 3.4 Mrad.

: 2 1 69478 .

Wog~l236t6 PC~S9~/0283 3~ .
T. results of Example 13 E-beam and gamma sterilized bovine carotid artery are detailed in Tables 8 and g below;
T~ble8: G~nmas~ d(3.l-3~4~&ad) Cu~dA~o~Ti~ue-(0.05M T~) T, (-C) SC~ial NO.solutioD Control S~
91-197-32 TniS 84.2 80.4 3.8 91-191-33 Tris 84.2 . 81.0 3.2 91-197-50 6~1iDC 84.0 79.8 4.2 91-197-86 ~ris 84.6 82.2 2.4 91-197-92 s~ e 84.6 79.8 4.8 ~ ~ ~ _~
~ ~ ~ 3~
M~ 84.3 79.8 4.5 1~1 S~lioe +0.4 10.0 +0.4 ~ ~ I 1~1111 n=2 n=2 1~=2 ~ ~11 ~ ~ 1~ ~!1 MC~D 84.3 81.2 3.1 ~ ¦ ~ 1 ~ I Tns ~0 2 +0 9 +0 7 " 2 1 69478 . .

WO 9Sn3616 PCIIUS9~/0283 T;lble 9. T,: E-Bcam (1.25 Mrad) Carotid Arl~ Tissue - (O.OSM Tris) T, ~C
Scri~l No.SOlU~JODCo~trol S~ "
gl-197-53ASaline 83.0 80.0 3.0 91-197-53BS~line 80.0 91-197-75ASalinc 84.6 83.0 1.6 91-197-75BS~liDe 82.0 91 19741A Tns 84.4 83.0 1.4 91-lg741B Tns NIA
91-197-72A Tris 84.6 83.0 1.6 91-197-72B Tris 83.0 91-197-79A Tns 81.4 83.0 -1.6 91-lg7-79B Tns 83.0 ~_ __.
il~ Mcan 83.8 81.3 2.3 çi c æ ~
c Z Saline +1.1 +1.5 +1.0 D=2 n=4 n=2 1~ Y~S m ~1 _ ~ ~ ~ ~ Mesn 83.5 83.0 0.5 Tns +1.8 +0.0 +1.8 ~ ~ ~ ~ ~ D=3 n=5 n=3 20 ~

Wog~l23616 - PCT~S9~/0283 T, data from E-beam and gamma irradiated bovine pericardium is summari2ed in Tables 10 and 11 below.

T~b~10. T.: ~BbunS~Gud(1~3~ Ebnie S 1.' ~''T~ooe T,~~
S~l~e 0.05M lns Control c. 1;,~ Cont~ol St~
Mca~l 81 81 81 81 Std. 2 Dev.
n= 14 21 15 3Z
Me~ ~ ~S ~ 0 13 ~ ~ ~ ~ O
~fier 1.2~ ~ i~

2 0 1~ ' ` T~ue T.(oC) - Saline O.O5M Tris Control Sterilized ControlSterilized Mean 81.4 78.6 81.~ 79.2 2 5 S~. 2.2 0.9 1.5 1. 1 Dev.
n= 14 28 15 30 Mean ~ 2.8 ~ 2.5 ExamPle 14. Approximately fifty bovine carotid artery grafts were processed under standard operating proced~res, except that half of the arteries were stored in saline rather than water i~mediately after harvesting.

21 6q478 - WO95~3616 PCT~59~/0283 Prior to fixa~ion, the grafts were stretched 45~ over their unstretched lengths. Fixation included a 24-hour ~xposure to 50 m~ citrate-buffered 0.l~ glutaraldehyde followed by 4.5 hours in citrate-buffered 2~
glutaraldehyde. The glutaraldehyde ~as drained from the fixation tank and replaced with 20 litres of RO-purified water to remo~e bulk excess glutaraldehyde and allowed to sit for approxi~ately 20 minutes. The entire volume of water was then replaced vith fresh water and allowed to sit for 23 hours for further diffusion of gl~taraldehyde from the graft tissue. The wa~er was then drained and filled with two more 20-litre aliguots of water and allowed to diffuse for approximately 20 more hours. At this point the water was replaced one final time prior to pa~kaging.
Grafts originally stored in water and grafts originally stored in saline ~ere evenly divided among three test groups. Those test groups were identified by various storage solutions: o.s~ sodium chloride, 0. 05M
Tris in o.s% sodium chloride adjusted to pH 7.4, and 0.lM
Tris adjusted to pH 7.4. The grafts were packaged in glass ~ials on glass mandrels and capped with the standard silicone-lined cips.
The grafts in each group were evenly divided into subgroups. One half of the grafts were exposed to gamma radiation with a dose ranging fro~ 2.02 to 2.24 Mrad.
The other half were E-beam sterilized at a d~se ranging from 2.43 to 2.55 Mrad. The grafts were tested for t~e following characteristics; radial tensile strength, suture retention strength, and T,. Tissue samples were also removed for histological evaluation using Masson Trichrome, Hematoxylin and Eosin, ~erhoeff's Elastica staining. Solution sa~ples were removed from each unit for determination of pH (before and after irradiation), osmolality, and glutaraldehyde content.
The grafts that were gamma sterilized were found to be somewhat discolored. Externally, the adventitial 21 6q478 WO 9SJ23616 ~ PC~ S9~10283 ~2 surfaces appeared grayish in color. A small number of grafts excised longitudinally revealed a grayish-purple lumenal aspect. No structural changes in the produce were apparent, however. Critical Surface Tension (CST) analysis was performed on six of the products (two from each of the three storage solutions) to determine ~hether the discoloration was caused by constitutional changes at the ~olecular level on the lumenal surface.
The quickest and most sensitive method of obtaining this information is by eva~uating the ~ettability of the surfaces, which may be determined ~y measuring liquid drop contact angle. Molecules deeper than 5-10 A from the surface have a negligible effect on surface/liquid interactions, so therefore, the contact angle is determined only by forces contributed by surface molecules. The contact angle is dictated by the balance of cohesi~e forces in the drop trying to curl it into a ball and ~dhesive forces between the liquid and the solid surface trying to cause the drop to spread.
CST is visualized by creating a Zisman plot, in which the cosines of contact angles of a series of pure alkanes are plotted against the surface tensions of the various liquids. A linear regression may be obtained by plotting this data. This CST is defined as the value on the surface tension axis that corresponds to cosine e (or contact angle - 0) for that particular surface.
Liquids that have surface tensions below the resulting cS~ will wet the surface and liquids with surface tensions greater than the CST ~ill yield observable contact angles.
Con~act angles ~ere measured using a Rame-hart goniometer per Inspection Procedure 690028. The fluids used in the analysis of the biological qrdft material were diiodomethane, b~omo-naphthalene, methyl-naphthalene, and hexadecane. CST values for the surfacestested were obtained by plotting the cosines of the observed contact angles against the surface tensions of :
W095/2361G ~CT~S9alO283 the four test fluids and extrapolating the resulting line to eosine e - 1. The x-~alue at that point is defined as the CST.

- 5 Results of the CST analysis are displayed in Table 12 below:
T~e ~ csr r- - S~ B~ Cs~id ~T~

S~npleNumb~ CST(~C)(d~n~/on) 91-29511S ~ 4 91-295~7T ~.6 91-2sS~4T 26.4 91-295-58ST 2~.1 91-295-81ST ~.7 Mn 26.
S~ on o.5 CST testing on both glutaraldehyde crosslinked bovine carotid artery and median artery tissue in the past has consistently yielded results in the range of 24-30 dynes/cm. The data above suggests no differences from non-irradiated tissue processed similarly in the past, as all ~C values lie well within ~he nor~al historical range. The use of CST to predict blood/surface interactions regarding thrombogenicity is not possible as there exists many mechanical and biological factors outside the real~ of interfacial chemistry that significantly effect t~rombotic activity. The test method was used only to detect deviations in surface molecular composition after irradiation from typical graft tissue.

`` 21 69478 woss/236l6 PCT~S95/0283 Results of T, testing performed on E-beam and gamma sterilized carotid artery graft tissue from Example 14 are summari2ed in Tables 13 and 14 below.
Table 13. T, of E-Beam Stcnlized (2.U - 2.55 MAd Carotid Arter~ Product T. (C) S~line O.lM Tris S~ e/O OSM T~is Control E-Beam CODtn;~l E-Be~m Control E-Beam Me~ 83 79 83 80 82 80 Std. Dev. 1 2 10 1~' 5 5 S 10 s 9 Me~ ~ ~ 4 ~a 3 ~ 2 2.43-2.55 ~ ~ ~ I ~1 i~ ~ ~1 ~, ~ ~ ~ ~ ~ ~ ~ ~ ~ Z~
F~ ~ 1~ 15~ ~ ~ ~ ~ ~

T~ 1~ T. o~ 2~d) C~:tid T,~C) Sal~ne 0.11~ Tns SaliDelO.OSM Tris Co~lrol Gamm~ Con~ol Gamma Coctrol Gamma MeaD 83 80 83 81 82 81 25 Std. Dev.
D= S 4 5 9 4 8 Ma~a ~ 3 ~ z 2.02-2.24 F~l~ a~ ~ ~ ~3 3 oMn~

pH of storage solutions before and after irradiation was tested and those results are summarized in Table 15 below. (Determination of pH prior to irradiation was perfor~ed on stock solutions rather than ` 2169478 ..
, W09~t23616 PCT~S9~to2835 for indlvidual graft units. Therefore, n=~ for all pre-sterili2ation samples).

Table 15. pH of Stor~ge S~ tS ~e- and Post-In~l ~t ~r~

J E-Be rn (2.~3-2.55Mrad) Gamma t2.02-2.24 ~d) Solution ~e Post Pre Post Saline 5.95 6.52 5.956.35 i 0.11 ~ o,oS
n=6 n--4 O.IM Tris 7.40 7.18 7.407.27 0.03 ~ 0.05 n--lO D=10 10Sal~el 7.40 6.98 î.40 î.06 O.O5M Tris + 0.0~ ~ 0.06 D--¦O D=10 ~pH ~alues for solutions _ ~ Tris m~y be sc~ t .uacc~ c as it ~as since beec lli~o~e,~J thlt a special glass calomel eltrode is r~quired for .~ P~C~ g Tris.

.
` 21 69478 , , WO 95~23616 ~CTtUS9~10283 Results of physical tests perfo~med on E-beam sterilized product f~om Example 14 is summarized in Table 16 below.

Table 16. ~h~sical Testin~ Results - E-beam S- " ~ Product (2.43 - 2.~ Mrad) W~ll Thickn~isSaline U~ ~ns0.05~ TristSaline (m n) 10Mean 0.94 1.11 0.99 Std.De~. 0.18 0.04 0.17 n= 108 189 171 Strengtb (Ibs) _ _ 15Mean 4.29 4.19 4.77 Std. De~. 1.22 0.90 1.12 n= 18 30 30 Suture ~
Strength (lbs) 20Me~n 2.18 2.14 2.05 Std. D~. 0.55 0.87 0.52 n= 18 36 27 , WO95/23616 PCT~S9~J0283 Results of physical tests performed on ga~ma sterilized product from Example 14 summarized in Table 17 below.

Tsble 1~ slcal Testin8 Results - Gamma Sterilized Product (2.02 - 2.~ Mr~d) W~l T~dn~s S~ine 0.1~ Tns 0.05M Tns/S~ine 10 (mm) Mean 0.94 l 00 o.g9 .
Std. De~. 0.25 0.18 0.17 n= 72 162 - 19~
~ _~ ~ ~ ~ ~ ~ ~_ ~
Ra~ial Ta~sile ~ ~ ~ i~EII ~ ~ ll ~ ~ ~ ~ ~! i~ lll 15Streogth (Ibs)~ ~ ~ ~ ~ ~ ~ iE~ ~ ~ ~ ~ l ~@
Mean 4.32 ~.4~ 4.39 Std. De~. 0.72 0.88 1.72 n= 12 27 33 20Stre~th(Ibs) Mo~ 1.83 1.91 2.02 Std.Dk~. 0.43 0.72 0.s7 n= 12 27 33 `: 21 69478 s W095/23616 PCT~S9~/0283 ~8 Osmolality of solutions samples post-E-beam are summarized in Table 18 below.

T~b~18. Pu~-E~Beu~ S~ P~d~Sd~ ~- -~ ~ r ~ l~ iD m~) ¦ S~ine O.IM Tns Sa1~ctO.OSM Tns Mu~ 278 156 3 S~.Dcv. 19 5 9 D~ 3 8 8 Residual glutaraldehyde le~els are summarized in Table l9 below.

Table19. Residu~ Glu~r~d~deLe~ds R~idu~ ~h~e~m) E-Beun (2.43 -2.55Mn~) Gamm~(2.02-2.24M~) Sal~e O.O5MSatlDe/ Sali~e 0.05MSaliDeJ
Tns0. IM Tns Tris 0. IM
Tns M~ 0-00 14.18 7.9~ l.Oo 10.397.38 25Std. Pev. NIA 2.î3 2.00 0.37 2.38 1.93 D= I2 20 20 4 I0 lO

._ Wo9~n36lG PCT~S9.~10~3 Results (~xa~ple 14):
1. Results of CST testing suggest there were no conformational deviations on the lumenal su~faces of the S irradiated products that were accountable for t~e noted discoloration.

2. T, data for E-beam sterilized carotid artery product suggest that the least amount of change resulted from packaging the tissue in the saline/0.05M Tris solution, follo~ed by O.lM Tris and saline. The same trend was noted for gamma sterilized product. The mean ~, of the E-beam sterilized product was approximately 2C less than the control material, while the T, for the gamma sterilized product was approximately 1C lower than the control.

3. Physical test results (radial tensile, suture retention, and ~all thic~ness) of E-beam and gamma sterilized graft product appear to be comparable to the current product, with and witho~t the use of Tris as a packaging solution additive.

4. of the two buffered storage solutions used in this 2~ example, the O.lM Tris appeared to have the greater buffering capacity: a decrease of 0.22 pH units compared to 0.42 with the saline/0.05M Tris combination following exposure to E-beam, and a decrease of 0.13 for O.lM Tris compared to o.34 for saline/0.05M Tris following gamma radiation. Based upon the superior buffering capacity o~
the more concentrated buffer, a saline/0. lM Tris storage solution ~as implemented in Example 15. It was discovered after these measurements were determined that a glass calomel electrode was necessary for testing pH of solutions containing Tris. The values, therefore, may be inaccurate.

2 ~ 69478 WO95~23616 ~CT~S9~J0283 5. The osmolality of the storage solutions was analyzed to gain some understanding of the tonicity of the various solutions used in these studies. It is advisable to maintain a near-physiological osmolality to prevent excessive swelling or shrinking of cellular components in the graft ~all which may contribu~e to stress on the collagen ~atrix. The final concentration of solute in - the packaging solu~ion may be adjusted to approach p~ysiological ~alues.
6. Residual glutaraldehyde analysis re~ealed a significant increase in concentration of glutaraldehyde, or another compound with the identical retention time under HPLC, fol~o~ing ~oth E-beam and gamma radia~ion.
The identity or origin of the peak has ~ot yet been deter~ined.

Example 15. Thirty bovine carotid artery grafts were placed on g~ass mandrels and stretched to 120~ of the~r incoming lengths (stretch ratio method) and placed in a 50m~ citrate buffered 0.1~-glutaraldehyde solution for a period of 24 hours. The graf~s were then pre-sterilized in a 50m~ citrate buffered 2~ glutaraldehyde solution for approximately 4 hours. ~ollowing sterilization, the grafts were rinsed with water: three fixa~ion tank volumes over a period of four days.
The graf~s were packaged in glass vials in one of two packaging solutions: saline or o.lM Tris brou~ht up in saline. The p~ of the saline/Tris solution was adjusted to 7.4 prior to packaging.
The test groups vere divided into two groups. One group was exposed to gamma radiation with a dose ranging from 2 . 5-2. 6 Mrad. The other half was ~-~eam sterilized at a dose cf 2.6 ~rad. The irradiated grafts were evaluated as follows: radial tensile strength, suture retention strength, and T,. Solution samples were removed 2 1 6~478 -WO9Sn36l~ PC~S9510283 from each unit for determina~ion of pH (before and after irradiation~, osmolality, and glutaraldehyde content.
Results of T, for Example 15 E-beam sterilized product are summarized in Table 20 below.

Table20. T.~ ~B~mS~ " ~6~Y~ Cun~d~oq ~ndu~
T,(C) Saline Saline/~.lM Tns Control E-Bcam Cootr~lE-Beam Me~n 84 78 83 79 Std. Dev.
n= 6 6 6 6 Mc~ ~a~ 6 ~ ~ ~ ~ 4 15 ~er~ ! l ~3 ~ E~ ~ l ~ , ~ .
2.6 Mt~ ~ ~ ~ ~ ~i ~a ~ ~ ~ l 7~ ! ~

Results of T, for Example 15 gamma ste~ilized produc~ is summarized in Table 21 below.
Table21. T,ofGammaSt~"~~~(2.5-2.C~d)C~o~dA~yP~d~t T,('C~
Saline SalinelO.lM Tris 2 5 Co~trol Gamma Con~l Gamma ~leao 84 80 83 81 Std. De~. 1 0.4 n= 6 6 6 ' 6 Me9n ~ 2Rcr ~1~ 4 ~ 2 2.6 Mrad -WO9~1t3616 PCT~S9;~n283' Results of physical tests performed on gamma sterili2ed product from Example 15 are sum~arized in Table 22 below.

5T~bk ~. n~ Te~ s~ GuD~a ~ " ' ~ t ~.S-2.~u~

Wsll Thickne~sSalineO.lM Tns/Saline ~mm) Mo~ 1.~ 1.03 Std. ~. 0.20 0.19 n= 108 108 ~ ~ ~ _~ b~
R~T~le Stn~thn~) M~ 4.32 3.67 Std.Db~. 1.22 1.03 n= 18 18 ~ ~ ~--~ ~ ~ ~ ~ ~ ~ ~ ~
sut~ n Strl!ngth(lbs) E I ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
20 Mo~ 2.31 2.13 Std.D~. 0.89 0.64 n= 18 18 . . , wossr236l6 PCT~S9~;/0283 Results of ph~sical tests performed on E-beam sterilized product from Example 15 are summarized in Table 23 below:

S T~ ~ n~sk~Ted~gRo~-~mS~ 'Pn~hLt ~.6M~u~

W~ hidcn~sS~lineO.lM Tns/Saline (mm) Mean 1.01 0.95 Std. De~. 0.20 0.18 nc 108 108 Rsdial Te~ile Stn:llg~ (Ibs) _~1 ~ ~ ~ 3 15 Me8n 4.49 3.99 Std. De~. 0.84 o.gg n= 18 18 Suture r,-St ~ n~) _ 20Mean 2.44 2.20 Std. De~. 0.68 0.66 n= 18 18 ` _ 2169478 Wo 95Q3616 PcrrUS95~0283 Residual glutaraldehyde results are detailed in Table 2 4 belo~.

T~blc24 R~id~lC`I ~ ~d'ydeL~vcls Residual GlutaraStehyde Levels am t2.6 M~) Gamma t2.5-2 6 Mrad~
S~l~nc Sal~e/Tns S~lineSalineJTris 2.25 9.83 0.43 10.19 1.82 9.60 0.54 13.52 1.69 l0.94 0.42 8.96 1.69 10.90 0.74 13.70 1.83 9.50 0.27 12.71 }5 1.91 10.13 ~i - 12.65 _~
0.00 12.16 ~ ~ ~ ~ ~ ~ 1 ~ ~ ~ ~ ~ ~ l ~ ~ ~
1.4~ 7.94 ~ 1.48 g.l9 aD ~ ~ ~ ~
2.~ 8.49 2.46 8.53 M~n: 1.55 9.91 0.48 11.82 Std.Dn.:0.78 1.53 0.17 2.12 Results:
1. T, results of ~oth gamma and E-beam sterilized product appear to be equivalent regardless of packaging solution, saline or Tris/saline.

2. Wall thickness values were comparable for all test groups.

`-- 2 1 69478 .i. . .

Wos~n3C16 PCT~S9~;/0283 3. Radial tensile strength of tissue stored in saline ~as somewhat greater for both methods of irradiation: 13%
greater for E-beam and 18~ greater for ga~ma. Tensile strength results of product stored in saline were slightly higher for product sterilized with E-beam: 4.4g lbs for E-beam and 4.32 lbs for gamma.

4. Similarly, suture retention streng~h of tissue stored in saline was greater for both methods o~ irradiation:
11~ greater for E-beam and 8% greater for gamma. Suture retention results of product stored in saline were sl~ghtly higher for product sterilized with E-bea~: 22.4 lbs for E-beam and 2.31 lbs for ga~ma.

5. As in Example 14, residual glutaraldehyde analysis revealed a significant increase in concentration of a compound with the identical retention time as glutaraldehyde under HPLC, following both E-beam and gamma radiation.
Example 16. ~icrobiologic~l Considerations. A
study was performed involving the E-beam sterilization of median artery tissue inoculated with Bacillus pumilus (0.5 - 5.0 x 106 spores per package). ~en samples were irradiated at 2.5 Mrad. There was no bacterial colonization present from any of the ten test samples after a fourteen day incubation period. When test results suggested a decrease in T, a~ter a dose of z.5 Mrad, a fGllow-up study was performed to determine the lowest dose that ~ould result in negati~e sterility ~esting with B . pumilus . Dose rates of 0.6 and 1.25 were applied to tissue inoculated with B. pumilus as described above. ~he samples irradiated at 1.25 Mrad exhibited a 100% kill rate while the mean T, of group (n = 5) was 3s 80.2C.

21 6q478 Wo95/2361G PCT~s9i/0~3 Example 17. The example determines the effectiveness of electron beam sterilization on biological graft tissue inoculated with a radiation-resistant organism.
Twenty-eight bo~ine median arteries were prepared SoP (stripped, ficin digested, and glutaraldehyde crosslinked~. Grafts ~ere aseptically packaged in glass ~ials in sterile saline and allowed to remain on the shelf for a period of 14 days to allow diffusion of excess glutaraldehyde from the tissue.
Twelve graf~s were removed from their respective vials and placed into a container with 7 liters of sterile saline (583 ml per unit) and allowed to soak for a period of 60 minutes to further diffuse residual glutaraldehyde.` Eac~ graft was placed in~o a foil laminate E-beam sterilization pouch~ Into each pouch, 150 ml steri7e saline was added and each was inoculated wit~ the following:
0.1 ml of Bacillus pumilus 0.5 to S.O x 106 spores/O.1 ml (organism indicated for radiation) Upon co~pletion of the packaging pro~ess, one pouch was omitted from the group intended for E-beam exposure ~o serve as a positive control. Test samples Z5 were then sterilized using E-beam radiation.
Ten out o~ ten test samples exhibited no bacterial coloni2ation after a fourteen day incubation period. The positive control sample exhibited bacterial colonization.
Electron beam sterilization was effective 7 n sterilizing ' 30 loO~ (10/10) of bovine-derived biological graft products packaged in saline and inoculated with a known radiation-resistant organism.

Example 18. This example tests the effects of electron beam sterilization on the physical properties of biological graft material. The following parameters were evaluated:

` 2169478 W09~123616 PCT~S9~Jo283 Radial Tensile Strength Suture Retention ~- Leak Rate Bursting Strength Shrink Temperature Critical Surface Tension - Histological Sectioning Twenty-eight b~vine median arteries were stripped, tied, sutured, digested, and glutaraldehyde fixed. The grafts were subjected ~o standard glutaraldehyde reduction steps to reduce glutaraldehyde residuals.
Grafts were packaged in 150 ml sterile 5aline in polyethylene pouches and heat sealed. Sterility and LAL
}5 samples ~ere ta~en a~ the repacking step to assure ~hat product being submitted for testing was sterile at the time of packaging. Samples were then exposed to E-beam radiation to sterilize the pac~aged arteries.
Results of physical testing is displayed in Table 25 below.

T~k ~.

TCS! E-Beam Control (mcan, s~d) (mean, std) RaLlia1 Tensilc (Ibs) 2.52 3.12 1.23 O.g2 Suhlre Retentiol~ (lbs) 0.88 1.19 0.37 0.38 Wsll Thiclc~ess (mm) 0.9~ 1.03 0.18 0.24 Lcatc Test (mlIm~ute) 2.0 2.3 1.4 4.5 Bursting Strength (psi) ~.0 61.0 19.3 Zl.7 3 0 S~sini; Tc-~ (C) 77.4 83.4 '0.2 ~. I
Critical Surf~lce Tcnsion 2~.t 26.
(~C, d~es/cm) 0.8 1.3 - ` - 2 1 69478 woss/236l6 PCT~S95/0283 While the invention has been described in some detail by way of illustration and example, it should be understood that the invention is susceptible to various ~odifications and alte~native forms, and is not restricted to the specific embodiments set forth. ~t should be understood that these specific embodiments are not intended to li~it the invention but on the contrary, the intention is to cover all modifications, equivalents, lo and alternatives falling within the spirit and scope of the invention.

Claims (17)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:-

1. A method for treating a biological tissue comprising exposing a biological tissue to a beam of accelerated electrons.

2. The method of claim 1 wherein irradiating the tissue comprises sterilizing the tissue.

3. The method of claim 1 including crosslinking said tissue.

4. The method of claim 3 including crosslinking said tissue with glutaraldehyde.

5. The method of claim 4 including placing the crosslinked tissue in a container prior to irradiation.

6. The method of claim 1 including irradiating the tissue with a sterilizing dose of E-beam particles.

7. The method of claim 6 wherein the sterilizing dose of E-beam particles is about 25 kGy.

8. A sterilized biological tissue comprising a tissue irradiated with a beam of accelerated electrons.

9. The tissue of claim 6 wherein said tissue is crosslinked.

10. The tissue of claim 9 wherein said crosslinked tissue has been crosslinked by glutaraldehyde.

11. A packaged sterilized biological tissue comprising a crosslinked biological tissue sealed in a container and exposed to E-beam irradiation.

12. A biological tissue for implantation comprising a sterilized biological tissue having decreased antigenicity and decreased polymeric degradation.

13. A biological tissue for implantation comprising sterilized biological tissue having improved performance characteristics.

14. The biological tissue of claim 13 wherein improved performance characteristics includes at least one of the following: softer, greater kink resistance, improved suturability, greater tensile strength, no loss of tensile strength, and increased effective orifice area, decreased pressure gradients and enhanced durability.

15. The biological tissue of claim 13 wherein the biological tissue is a heart valve, vascular prosthesis, or pericardial patch.

16. The method of claim 1 wherein the biological tissue is a heart valve, vascular prosthesis, or pericardial patch.

17. The method of claim 2 wherein sterilizing the tissue comprises terminal sterilization.