Patent Publication Number: US-6210957-B1

Title: Apparatuses for treating biological tissue to mitigate calcification

Description:
RELATED APPLICATIONS 
     The present application is a divisional of U.S. application Ser. No. 08/874,180, filed Jun. 13, 1997, now U.S. Pat. No. 5,931,969 entitled “Methods and Apparatuses for Treating Biological Tissue to Mitigate Calcification”, which is a continuation of U.S. application Ser. No. 08/282,358, filed on Jul. 29, 1994, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention pertains generally to apparatuses for preparing biomedical materials, and more particularly to an apparatus for preparing preserved biological tissue, such as bovine pericardium, for implantation in a mammalian body that induces relative treatment fluid/tissue motion. 
     BACKGROUND OF THE INVENTION 
     The prior art has included numerous methods for preserving or fixing biological tissues, to enable such tissues to be subsequently implanted into mammalian bodies. Examples of the types of biological tissues that have heretofore been utilized for surgical implantation include cardiac valves, vascular tissue, skin, dura mater, pericardium, ligaments and tendons. 
     The term “grafting” as used herein is defined as the implanting or transplanting of any living tissue or organ (See Dorlands Illustrated Medical Dictionary, 27th Edition, W. B. Saunders Co. 1988). Biological tissues which are grafted into the body of a mammal may be xenogeneic (i.e., a xenograft) or allogeneic (i.e., an allograft). The term “bioprosthesis” defines many types of biological tissues chemically pretreated before implantation (Carpentier—See Ionescu (editor), Biological Tissue in Heart Valve Replacement, Butterworths, 1972). As opposed to a graft, the fate of a bioprosthesis is based upon the stability of the chemically treated biological material and not upon cell viability or host cell ingrowth. Chemical pretreatment includes the “fixing” or tamning of the biological tissue. Such fixing or tanning of the tissue is accomplished by exposing the tissue to one or more chemical compounds capable of cross-linking collagen molecules within the tissue. 
     Various chemical compounds have been utilized to fix or cross-link biological tissues including formaldehyde, glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate and certain polyepoxy compounds. 
     In particular, glutaraldehyde has proven to be relatively physiologically inert and suitable for fixing various biological tissues for subsequent surgical implantation (Carpentier, A., J. Thorac. Cardiovasc. Surg. 58:467-68 (1969)). In particular, examples of the types of biological tissues which have heretofore been subjected to glutaraldehyde fixation include porcine bioprosthetic heart valves and bovine pericardial tissues. 
     Clinical experience has revealed that glutaraldehyde-fixed bioprosthetic tissues may tend to become calcified. Such calcification of glutaraldehyde-fixed bioprosthetic tissues has been reported to occur most predominantly in pediatric patients see, Carpentier et al. and “Continuing Improvements in Valvular Bioprostheses, J. Thorac Cardiovasc. Surg. 83:27-42, 1982. Such calcification is undesirable in that it may result in deterioration of the mechanical properties of the tissue and/or tissue failure. In view of this, surgeons have opted to implant mechanical cardiovascular valves into pediatric patients, rather than to utilize glutaraldehyde-preserved porcine valves. However, pediatric patients who receive mechanical valve implants require long term treatment with anticoagulant medications and such anticoagulation is associated with increased risk of hemorrhage. 
     The mechanism by which calcification occurs in glutaraldehyde-fixed bioprosthetic tissue has not been fully elucidated. However, factors which have been thought to influence the rate of calcification include: 
     a) patient&#39;s age 
     b) existing metabolic disorders (i.e., hypercalcemia, diabetes, kidney failure . . . ) 
     c) dietary factors 
     d) race 
     e) infection 
     f) parenteral calcium administration 
     g) dehydration 
     h) distortion/mechanical factors 
     i) inadequate coagulation therapy during initial period following surgical implantation; and 
     j) host tissue chemistry 
     Methods for treating fixed biological tissue so as to inhibit calcification thereof following implantation in a mammalian body tend to substantially increase the usable life of such tissue subsequent to implantation in a mammalian body, thereby mitigating the requirement for subsequent tissue replacement. As those skilled in the art will appreciate, such tissue replacement frequently causes substantial trauma to the patient, occasionally resulting in the patient&#39;s death. As such, it is greatly beneficial to be able to either avoid or postpone the need for the replacement of implanted biological tissue. 
     Various efforts have been undertaken to find ways of mitigating calcification of glutaraldehyde fixed bioprosthetic tissue. Included among these calcification mitigation techniques are the methods described in U.S. Pat. No. 4,885,005 (Nashef et al.) SURFACTANT TREATMENT OF IMPLANTABLE BIOLOGICAL TISSUE TO INHIBIT CALCIFICATION; U.S. Pat. No. 4,648,881 (Carpentier et al.) IMPLANTABLE BIOLOGICAL TISSUE AND PROCESS FOR PREPARATION THEREOF; U.S. Pat. No. 4,976,733 (Girardot) PREVENTION OF PROSTHESIS CALCIFICATION; U.S. Pat. No. 4,120,649 (Schechter) TRANSPLANTS; U.S. Pat. No. 5,002,2566 (Carpentier) CALCIFICATION MITIGATION OF BIOPROSTHETIC IMPLANTS; EP 103947A2 (Pollock et al.) METHOD FOR INHIBITING MINERALIZATION OF NATURAL TISSUE DURING IMPLANTATION; WO84/01879 (Nashef et al.) SURFACTANT TREATMENT OF IMPLANTABLE BIOLOGICAL TISSUE TO INHIBIT CALCIFICATION; U.S. Pat. No. 5,595,571 (Jaffe) BIOLOGICAL MATERIAL PRE-FIXATION TREATMENT; and WO95/11047 (Levy et. al.) METHOD OF MAKING CALCIFICATION-RESISTANT BIOPROSTHETIC TISSUE. 
     Although some researchers believe that glutaraldehyde actually increases the risk of calcification, it is still the most accepted fixation solution. For example, the Levy patent application noted above utilizes an alcohol treatment for mitigating calcification, in addition to a glutaraldehyde fixation 
     There is significant research occurring into the extent the mechanisms mentioned above cause calcification. Many processes are believed to mitigate calcification, without their proponents knowing exactly why. Indeed, the Levy patent does not offer a mechanism why alcohol is effective in calcification mitigation, other than it is preferred over aldehydes. 
     A number of tests are conventionally used to gauge the efficacy of various calcification mitigation treatments. The most reliable test is actual implantation into a living organism, preferably a human. Of course, such host studies are by their nature long-term and the results somewhat skewed by the variations present in each individual host. Researchers are therefore constrained to predict the ultimate calcification mitigation benefits of a particular treatment by using laboratory tests on treated tissue, such as calcium uptake studies. Ultimately, there is a substantial amount of extrapolation from the empirical data of such laboratory tests, and to date there is no one predominant mechanism recognized for mitigating calcification. 
     There remains a need for the development of new methods for inhibiting or mitigating calcification of chemically fixed biological tissue. 
     SUMMARY OF THE INVENTION 
     These, as well as other advantages of the present invention will be more apparent from the following description and drawings. It is understood that changes in the specific structure shown and the described may be made within the scope of the claims without departing from the spirit of the invention. 
     The present invention provides a method for treating at least partially fixed biological tissue to inhibit calcification of the tissue following implantation in a mammalian body, comprising immersing the tissue in a treatment solution, inducing relative and repeated tissue/solution movement, and beating the solution during the step of inducing. The step of inducing may comprise flowing treatment fluid across the tissue and restraining the immersed tissue from gross movement, or enclosing the treatment solution in a container and either shaking the container or stirring the solution within the container, with the immersed tissue floating free or being restrained from gross movement within the container. The step of heating may be applying beat to the outside of the container to indirectly heat the solution therein, or placing the treatment container in an enclosure and heating the enclosure. Alternatively, the step of heating may comprise applying heat directly to the treatment solution. 
     The present invention also includes a method for treating at least partially fixed biological tissue to inhibit calcification of the tissue following implantation in a mammalian body, comprising positioning the tissue in a flow container; restraining the tissue from gross movement within the container, flowing treatment solution through the flow container into contact with the tissue, and heating the solution during the step of flowing. The step of restraining may comprise mounting the tissue in a planar configuration substantially parallel to the direction of flow of the flowing solution. The tissue may be positioned within a flow container having a cross-section oriented substantially normal to the direction of flow of the flowing solution, the tissue being positioned downstream of a baffle to create a substantially uniform downstream flow profile over the cross-section. In one embodiment, treatment solution is supplied to an inlet of the flow container from a reservoir, and fluid is expelled from an outlet of the flow container to the reservoir. The treatment solution may be heated in the reservoir. Preferably, the treatment fluid flows upward through the flow container from the inlet to the outlet and into contact with the tissue. 
     In accordance with the invention, an apparatus for treating at least partially fixed biological tissue to inhibit calcification of the tissue following implantation in a mammalian body is provided. The apparatus comprises a flow container, a supply of treatment fluid, a fluid input to the container, a fluid output from the container, a tissue mount for positioning the at least partially fixed biological tissue within the container between the input and output and restrain its gross movement therein, and means for heating the fluid. The flow container is preferably divided into at least two sections in series separated by perforated baffles, with at least one tissue mount in each section. The flow container may be an elongated tube and the baffles circular. The tissue mount may be configured to mount the tissue in a planar configuration substantially parallel to the direction of flow of the solution flowing through the container. The apparatus may additionally include at least one baffle positioned in the flow container and upstream of the tissue mount, the baffle being configured to create a substantially uniform downstream flow profile over a cross-section of the flow container. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a flow diagram illustrating the prior art process for preparing biological tissue for implantation within a mammalian body comprising fixing of the biological tissue with a glutaraldehyde solution; 
     FIG. 2 is a flow chart of the preparation of biological tissue for implantation in a mammalian body comprising a method for inhibiting calcification of the biological tissue according to the present invention; 
     FIG. 3 is a schematic view of an exemplary tissue treatment apparatus including a closed treatment container and container movement device; 
     FIG. 4 is a schematic view of another exemplary tissue treatment apparatus including an open treatment container and fluid stirring rod; 
     FIG. 5 is a flow chart of the preparation of biological tissue using the system of FIG. 3 or  4  including the application of heat and motion to a treatment solution; 
     FIG. 6 is a schematic view of an exemplary tissue treatment apparatus including a treatment container positioned in a flow stream; 
     FIG. 7 is a flowchart of the preparation of biological tissue using the system of FIG. 5 including the application of heat and flow of treatment solution past the tissue; 
     FIG. 8 is a perspective view of another preferred tissue treatment apparatus including an upstanding flow column and a plurality of vertical sections within which tissues to be treated are mounted; 
     FIG. 9 is an enlarged perspective view of one vertical segment of the flow column of FIG. 8 illustrating a piece of tissue suspended from a baffle in a flow stream; 
     FIG. 10 is a horizontal cross section taken along line  10 — 10  of FIG.  9  through one vertical section of the flow column; 
     FIG. 11 is a vertical cross section taken along line  11 — 11  of FIG.  10  and through a baffle and tissue suspension mount; 
     FIG. 12 is a bar graph comparing the measured calcium uptake in bovine pericardium tissues treated in a conventional manner, solely with heat, and with heat and motion; and 
     FIG. 13 is a bar graph comparing the measured calcium uptake in bovine pericardium tissues treated in a conventional manner and with heat and motion from various sources. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The detailed description set forth below in connection with the appended drawings is intended as a description of the presently preferred embodiment of the invention, and is not intended to represent the only form in which the present invention may be constructed or utilized. The description sets forth the functions and sequence of steps for constructing and operating the invention in connection with the illustrated embodiment. It is to be understood, however, that the same or equivalent functions and sequences may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention. 
     One method for treating glutaraldehyde fixed biological tissue to inhibit calcification thereof following implantation in a mammalian body is illustrated in FIG. 2 which depicts a flow chart of the presently preferred embodiment of the invention. FIG. 1 depicts a flow chart of the prior art method for preparing biological tissue for implantation within a mammalian body. 
     Referring now to FIG. 1, the prior art process for preparing biological tissue for implantation within a mammalian body comprises first harvesting the tissue from an animal or human cadaver  10 . As those skilled in the art will recognize, various different types of tissue are routinely harvested from different animals and/or human cadavers. For example, heart valves are routinely harvested from pigs, pericardium is routinely harvested from cows or pigs, and skin is routinely harvested from human cadavers. Those skilled in the art will further recognize that new tissues are, from time to time, being found to be implantable within a mammalian body. 
     After harvesting, the biological tissue is rinsed in saline solution, typically for a period of 1-6 hours  12 . 
     The tissue is next fixed using a buffered glutaraldehyde solution of adequate concentration, for example between 0.2% and 0.8%, at room temperature for at least 3 hours  14 . As is well known, glutaraldehyde effects cross-linking of the proteins, e.g., collagen, within the tissue. Such cross-linking tends to make the tissue more durable and effects preservation thereof. It is known that cross-linked protein exhibits increased resistance to proteolytic cleavage and further that one of the major processes by which circulating blood may destroy tissue is via enzymatic activity which involves unfolding of the protein substrate in order to facilitate enzymatic hydrolysis. Cross-linking of the protein of a tissue makes the tissue resistant to such unfolding, and consequently tends to prevent deterioration thereof due to the enzymatic activity of blood. 
     The tissue is next sterilized, preferably with an alcohol/formaldehyde solution for 2 hours at room temperature 16. The preferred solution for effecting sterilization of the tissue comprises approximately 12 ml/l of Tween 80; approximately 2.65 gms/l of MgCl2.H2O; approximately 108 ml/l of formaldehyde (37%); approximately 220 ml/l of ethyl alcohol (100%) and approximately 4.863 gms/l of HEPES buffer. The balance of the solution comprises double filtered H2O. The pH of the solution is typically adjusted to 7.4 via the addition of NaOH. Those skilled in the art will recognize various other sterilization solutions are likewise suitable. 
     Antimineralization treatment  18  is optionally performed so as to inhibit the accumulation of mineral deposits upon the biological tissue after implantation of a mammalian body. As those skilled in the art will recognize, various different antimineralization treatments are utilized so as to prevent the deposition of various different minerals upon the biological tissue. 
     The tissue is trimmed and any non-biological components are then added thereto  20 . For example, it is common to sew a heart valve to a valve holder which aids in the handling thereof and which may additionally function as a mount for the valve when implanted into a mammalian body. 
     Next, the biological tissue is once again sterilized  22 , preferably in an alcohol/formaldehyde solution as discussed above. Since preparation of the biological tissue is substantially complete and the biological tissue will next likely be stored for an extended period of time, a more rigorous sterilization procedure from that previously utilized is typically employed. At this stage, the biological tissue is typically sterilized for approximately 9 hours at 34-38° C. 
     After sterilization, the biological tissue is stored in glutaraldehyde at room temperature 24. 
     Tissue Treatment Using Heat 
     Referring now to FIG. 2, a method for treating glutaraldehyde fixed biological tissue to inhibit calcification thereof following implantation in a mammalian body comprises the additional step of heating preferably when the glutaraldehyde is in contact with the biological tissue, to approximately 35-75° C. for approximately 4-22 weeks, and more preferably for a period of a few days to 22 weeks. 
     The treatment fluid should be heated to a temperature greater than body temperature (37° C.) but not high enough to damage either the tissue or the treatment fluid. Thus, the preferred heat range is between 35-75° C. However, the temperature affects the amount of calcification mitigation, and the process time, and is preferably between 45° C. and 55° C., and more preferably between 50° C.±1° C. 
     Heating of the biological tissue may be performed at any time after harvesting the tissue from the animal or human cadaver and prior to implanting the tissue within a mammalian body. However, heating of the biological tissue is preferably performed at a point in the process for preparing the biological tissue when the biological tissue is already disposed within a bath of glutaraldehyde solution, as occurs at various stages of the process according to the prior art. Thus, the method for treating glutaraldehyde fixed biological tissues according to the present invention is preferably performed either during fixing thereof with a glutaraldehyde solution, immediately after fixing thereof with the glutaraldehyde solution, or alternatively just prior to or after being stored in a glutaraldehyde solution. 
     As a further alternative, a method for treating glutaraldehyde fixed biological tissues may be performed during antimineralization treatment by adding glutaraldehyde to the antimineralization solution and heating the solution, preferably to approximately 35-75° C. for approximately 4-22 weeks. More preferably, the tissue is heat treated at 50° C.±1° C. for a period of a few days to 22 weeks. 
     For example, after fixing tissue using a buffered glutaraldehyde solution of adequate concentration, for example between 0.2% and 0.8%, at room temperature for at least 3 hours  14 , the biological tissue may be heat treated in either the same or different glutaraldehyde solution, preferably at approximately 35-75° C. for a few days to 22 weeks  15 . 
     As one of the alternatives discussed above, the biological tissue is fixed and heat-treated simultaneously  13  in the 0.2-0.8% glutaraldehyde solution, again preferably at approximately 35-75° C. for approximately a few days to 22 weeks. Another alternative is to heat the tissue in saline  17  prior to fixation  21 . 
     As the other alternative discussed above, the biological tissue may simultaneously undergo antimineralization treatment and heat treatment  19 . Glutaraldehyde is added to the antimineralization solution so as to effect the inhibition of calcification of the tissue following implantation in a mammalian body. 
     Tissue Treatment Using Relative Tissue/Fluid Movement 
     FIG. 3 illustrates one preferred embodiment of a tissue treatment system  20  of the present invention. One or more pieces of tissue  22  or leaflets are immersed in a treatment solution  24  within a closed container  25 . The container  25  rests on a shaker table  26  which reciprocates relative to a base  27  in one or more directions. One particularly preferred type of shaking device is an orbital shaker. In one exemplary embodiment, the orbital shaker  26  is actuated at a rotational speed of approximately 55 RPM. The container  25  and contents therein may be subjected to heating, such as with radiant heaters  28  as illustrated. Of course, any number of means for heating the container  25  are known, such as resistance heaters, convective flow, and the like. 
     The solution  24  is preferably a buffered glutaraldehyde, but may be any chemical solution, such as Denacol® or others, which performs substantially the same in this context. The shaking and/or heat may be applied during fixation or after. The tissue is preferably at least partially fixed prior to being subjected to the calcification mitigation treatment described herein, and more preferably the tissue is fully fixed prior to the treatment. The treatment thus can be designed to complete the fixation process. In a preferred embodiment, tissue that has been fixed for a period of between thirty minutes to fourteen days is placed in the container  25  with a buffered clutaraldehyde solution of adequate concentration, for example between 0.2% and 0.8%. The solution is then shaken for thirty minutes after which the container  25  remains static for fourteen days. 
     The tissue  22  may be sheets of bovine pericardium tissue, precut leaflets, or fully formed porcine heart valves. One potential disadvantage of using precut leaflets or porcine heart valves is the tissue&#39;s nonuniform capacity for shrinkage during calcification mitigation treatment. It can be difficult, though not impossible, to consistently and accurately compensate for this phenomenon. A detailed map of the fiber orientation, thickness and other properties of each individual leaflet may be required to predict the final form of the leaflet after treatment. Therefore, the preferred procedure is to place sheets or pieces of tissue in the container and subject it to the shaking and/or heat. Afterwards, the leaflets are cut from the treated tissue. 
     It will be noted that the tissue  22  within the solution  24  may be allowed to move about freely. In another embodiment, and as will be described below with respect to the embodiment of FIG. 6, the tissue may be restrained from gross movement but allowed to freely shrink, such as with a device schematically shown at  29 . 
     In another variation on the shaking, a treatment system  30  is shown in FIG. 4 wherein a stirring rod  32  is positioned in a container  34  to replace the shaking table  28 . The stirring rod is preferably actuated magnetically through the container, but may also comprise a shaft driven apparatus. The stirring rod  32  is preferably designed so as not to batter the tissue  36  but instead just to cause gentle movement of the fluid  37  relative to the tissue. Therefore, in the illustrated embodiment, a piece of filter paper  38 , or other such similar porous substrate or mesh, is draped over the top rim of the container and the tissue pieces  36  placed therein. In this way, the stirring rod  32  imparts rotational or other momentum to the fluid  37  in the container  34 , but the tissue  36  remains above the damaging action of the rotating rod. Also shown in FIG. 4 is a heated enclosure or incubator  39  within which is placed the entire apparatus  30 . 
     In another version of shaking, multiple flasks or containers holding the treatment fluid and tissue samples are clamped to a rotating ferris-wheel apparatus. The apparatus includes a wheel rotating about a tilted axis so that the flasks follow a tilted circular trajectory. In this manner, the fluid within the flasks gently washes over the tissue pieces as the wheel rotates. 
     The containers  25  and  34  in FIGS. 3 and 4 may be open or closed, primarily depending on the nature of the treatment fluid. Glutaraldehyde is a toxic substance which evaporates to create a dangerous gas. Thus, treatment with glutaraldehyde is preferably done in a closed container. On the other hand, some substances like Denacol® may be less hazardous and the container may be left open under a hood, for example. 
     Relative movement between the tissue and the treatment fluid is believed to enhance calcification mitigation. A mechanism for this result has not been fully formulated, although mass transport of the fluid surrounding the tissue may be relevant. Indeed, one theory is that certain cell material, for example, proteins, is extracted or removed from the tissue by the treatment fluid, which removal is enhanced relative to static treatment methods by the movement of the fluid. In other words, the relative movement of the tissue within the fluid repeatedly replenishes the fluid surrounding any one portion of tissue. Test results shown in FIGS. 12 and 13 for samples of tissue treated in a variety of ways in accordance with the present invention indicate that the combination of heat and relative tissue/fluid movement decreases the amount of calcium uptake after implantation in rats, suggesting that such treatment will mitigate calcification in long or short term implantation in humans. 
     FIG. 5 is a flowchart showing a preferred method for treating tissue using the system shown in FIGS. 3 or  4 . Many of the specific pre- and post-treatment steps described with respect to FIGS. 1 and 2 have been left out for clarity, but remain applicable. Initially, the tissue is harvested, rinsed, fixed and cut into pieces, preferably squares or rectangles, from which leaflets may be formed. The pieces of tissue are then immersed in the treatment fluid within the container, and the fluid heated to a predetermined temperature. Relative movement between the tissue pieces and surrounding treatment medium is induced and continued for a predetermined time. Inducing relative tissue/fluid movement may be accomplished by any of the configurations shown herein, such as shaking or vibrating a container for the tissue and fluid, or by flowing treatment fluid onto the tissue. Finally, the tissue pieces are removed from the container, rinsed and stored for later use. Of course, rather than storing the tissue, it may be formed directly into leaflets and assembled into a heart valve directly after the treatment process. 
     The solution is heated indirectly through the surrounding air, such as with the radiant heaters  28  shown in FIG. 3, to a temperature of about 50° C. plus or minus 1° C. The container is shaken or the fluid is stirred to cause relative tissue/fluid movement. The treatment time ranges between fourteen days to two months, but is preferably closer to two months. The container  25  is preferably a glass tissue culture flask having a volume of approximately 250 ml., and the solution is a buffered glutaraldehyde solution of adequate concentration, for example between 0.2% and 0.8%. As mentioned above, a number of pieces of tissue  22  may be treated at a single time within the container  25 . One proposed ratio of tissue to solution is approximately 12 leaflets or leaflet-sized pieces of tissue per every 150 ml of solution. 
     Tissue Treatment Using Relative Tissue/Fluid Flow 
     FIG. 6 illustrates schematically another variation on a treatment system  40  which utilizes flow past the tissue as opposed to shaking a container or stirring the fluid in which the tissue is placed. A flow creates the relative motion between the treatment solution and the tissue which is believed to result in the beneficial calcification mitigation effects. 
     The system  40  comprises a flow container  42  within which tissue  44  is placed. A number of conduits  46  connect one end of the flow container  42  to a pump  50  and then to a solution reservoir  48 . Conduit  47 , shown in dashed line, may be coimected between the other end of the flow container  42  and the reservoir  48  to complete a closed circulation loop. The pump propels treatment solution through the system  40  in the direction shown by the arrows  52 . The tissue  44  is preferably restrained within the flow container  42  using means schematically illustrated at  56 . Resistance heaters  54  are illustrated surrounding the reservoir  48 . If immersion heaters are used, they must be able to withstand the extended exposure to sometimes caustic treatment fluid. Of course, one or both of the resistance heating elements  54  may be removed from around the reservoir, or alternative heating devices may be used. For example, treatment system  40 , and the system  20  or  30  shown in FIGS. 3 and 4, for that matter, may be enclosed in a larger enclosure or room  58  which is heated to the preferred temperature by internal or external heaters. In the illustrated embodiment, thermocouples  59  are provided to sense the temperature within both the flow container  42  and the reservoir  48 . The thermocouple  59  in the reservoir is preferably connected to feedback electronics for controlling the heaters  56  based on the temperature of the fluid in the reservoir. This is so that the temperature does not rise too high to a level which might be detrimental to the tissue. The temperature within the flow container is monitored using a thermocouple both as a safety, and to record the precise temperature profile of the treatment fluid. 
     The basic elements of a method for treating tissue using the system  40  are illustrated in FIG.  7 . Initially, the tissue is harvested, rinsed, fixed and cut into pieces, preferably squares or rectangles, from which leaflets may be formed. The tissue (or leaflets in some instances) may be placed within the flow container  42  and subjected to flow during or after fixation. In a preferred embodiment, the tissue  44  is at least partially fixed before being subjected to the flow within the system  40 , and more preferably the tissue is fully fixed prior to the treatment. The pieces of tissue are then placed in the treatment container, and the solution caused to flow therethrough, initiating relative movement between the tissue pieces and surrounding treatment medium which is continued for a predetermined time. The solution is heated directly outside of the container, or indirectly by heating the container. Finally, the tissue pieces are removed from the container, rinsed and stored for later use. Of course, rather than storing the tissue, it may be formed directly into leaflets and assembled into a heart valve directly after the treatment process. 
     With reference to FIG. 6, the tissue is first fixed for a period of between thirty minutes to fourteen days and placed in the flow container  42 . In an alternative, the tissue may be first placed within the container  25  shown in FIG.  3  and shaken for a period of thirty minutes. After the fixation (or after the shaking, if desired), the tissue is placed in the flow container  42  and subjected to solution flow of between ten and fifteen gallons per minute (38-57 lpm) for a period of between fifteen to sixty days. The solution is preferably heated directly within the reservoir  48  to a temperature of about 50° C. (122° F.). The solution is preferably a 0.2-0.8% buffered glutaraldehyde, and the tissue  44  is restrained from movement but allowed to shrink. 
     In an alternative method of treating tissue in the system  40 , the treatment time is between thirty and sixty days. The flow rate is approximately 7.4 gallons per minute (28 lpm) on average, and is uniform throughout a cross section normal to the flow within the flow container  42 . The tissue  44  is preferably a rectangle of bovine pericardium of about 2 inches by 4 inches in dimension. This size of tissue sample may be used to form one or two leaflets after treatment. 
     Those with skill in the art will recognize that variations to the above mentioned systems and processes for moving the fluid and/or heating the tissue are available. For example, the flow of solution past the tissue may be combined with a vibrational or shaking motion of the flow container  42  to enhance any calcification mitigation benefits derived from either method. Additionally, though the system  40  is shown as a closed circulation device, fresh solution may be pumped to the flow container  42  and discharged after passing through the container (thus the conduit  47  is shown as optional). Of course, this will require a significant amount of treatment solution which may be prohibitively expensive. Nevertheless, one of the theoretical mechanisms for the beneficial aspects of the present treatment method including flow is that the solution is constantly replenished in the region surrounding the tissue so that a maximum mass transport of chemicals and/or biological material such as protein is realized from the tissue to the solution. Thus, a system which inputs fresh treatment solution, rather than recycling it through a reservoir, would theoretically be more effective in this regard. 
     Flow Column Apparatus 
     FIG. 8 illustrates a perspective view of a flow column  60  which may represent the flow container  42  illustrated schematically in FIG.  6 . The column  60  is preferably a clear acrylic tube  61  having an inner diameter of approximately six inches (15.2 cm), a height of about six feet (1.8 m), and a capacity of about ten gallons (38 l). The top and bottom ends of the cylinder  60  are closed by caps  62   a  and  62   b,  respectively, which are sealed against the inner surface of the cylinder  60  with O-rings (not shown). A lower inlet fitting  64  centered in the cap  62   b  provides a conduit for introducing treatment fluid to the lower end of the cylinder  60 . Likewise, an upper fitting  66  connected to the cap  62   a  provides an outlet for the treatment fluid. A length of hose  68  connects the lower fitting  64  to a fluid pump  70 , which is in turn connected by a hose  72  to a fluid reservoir  74 . A length of hose  76  connecting the upper fitting  66  to the reservoir  74  completes the circulatory treatment system. Those with skill in the art will understand the fluid connections and requirements, which will not be described further herein. 
     As mentioned above, the solution within the reservoir  74  is preferably directly heated to the desired treatment temperature. Although not illustrated, the reservoir is desirably provided with one or more immersion resistance heaters. A thermocouple  77  senses the temperature of the reservoir and is preferably connected to feedback electronics for controlling the immersion heater so that the solution temperature does not rise too high to a level which might be detrimental to the tissue. The temperature within the flow container is monitored using a thermocouple  78  both as a safety, and to record the precise temperature profile of the treatment fluid. Excessive temperatures can detrimentally affect the treatment solution itself, and thus the heating must be done gradually and with a heater having good temperature control. 
     The vertical flow column or cylinder  60  is segmented into a plurality of vertical sections  80  (seen enlarged in FIG. 9) by a number of regularly spaced baffles  82  having perforations  83 . The baffles are substantially circular perforated disks positioned horizontally within the vertical cylinder  60 , normal to the fluid flow. The outer diameter of each baffle  82  contacts, or comes into close proximity with, the inner surface of the tube  61 . Although the flow column  60  is illustrated vertically, other arrangements will work. However, the vertical flow orientation is preferred to help purge bubbles from the flow column at start up. In other words, the bubbles naturally migrate out of the flow column in a very short time, as opposed to a horizontal flow path, for example. It should be also be noted that the perforations are not shown in FIGS. 8 and 9 for clarity, but are shown in FIG.  10 . 
     The baffles  82  are commonly mounted on a vertical support rod  84  extending along the axis of the cylinder  60 . The support rod  84  contacts the lower ceiling cap  62   b  and extends upward into close proximity to the upper cap  62   a.  As seen at the lower end of FIG. 8, the support rod  84  preferably terminates in a stand member  86  having a pair of bifurcated legs  88  which contact the top surface of lower cap  62   b  on either side of an inlet aperture  90 . In this manner, the support rod  84  can be positioned along the axis of the cylinder  60  while not occluding inlet flow from the pump  70 . 
     As mentioned above, the baffles  82  divide the cylinder  60  into a plurality of vertical sections  80 . In this respect, the vertical sections  80  include the region between two baffles  82 . In the illustrated embodiment, there are eight such vertical sections  80  having a height of between seven and eight inches (17.8-20.3 cm). The entire height of the column  60  is approximately 6 feet (1.8 m), and thus there is some space left above the top baffle and below the bottom baffle. The baffles  82  are slidably mounted on the support rod  84  to enable adjustment of the spacing therebetween, if desired. Furthermore, the tissue pieces  82  can be easily mounted when the baffles  82  are removed from the system, whereupon the baffles are slid over the support rod which is then positioned within the tube  61 . The tissue pieces to be treated are mounted in a particular manner in a circumferential array about the support rod  84 , as will be apparent from the description of FIGS. 9-11. 
     At the top of the cylinder  60  a vertical space is created between the upper baffle and the upper cap  62   a,  in which the central support rod  84  terminates. The space is needed to insure that the flow passing through upper baffle  82  is not unduly disturbed so that the flow within the upper vertical section  80  remains uniform in a horizontal cross section. Indeed, the uniformity of flow across any horizontal cross section between the baffles is important in the present configuration to insure that the flow past any one piece of tissue is equal to the flow past other tissues. The primary mechanism for insuring such uniform flow is the baffles  82  themselves. Preferably, the perforations  83  are sufficiently numerous and have a sufficient diameter so that the cross-sectional area of the baffles  82  has less structural material than open flow channels. The baffles  82  are thus designed to maintain a uniform, non-laminar upward flow stream through each flow section  80 . 
     At the lower end of the cylinder  60 , below the lowest baffle  82 , a flow straightener  92  is positioned just above a velocity reducer plate  94 . Inlet flow through the aperture  90  thus passes upward through the velocity reducer plate  94  and flow straightener  92  to impinge on the lowest baffle  82 . The velocity reducer plate  94  is a disc like plate having a plurality of apertures  96  formed therein. The apertures are relatively widely spaced in the plate  94  to create a drag on the flow and slow its velocity. The flow straightener  92  resembles a honeycomb structure with a relatively densely spaced number of individual flow channels, and has a vertical dimension greater than the velocity reducer plate  94  or baffles  82 . Flow enters the column  60  through the aperture  90  and continues upward through the velocity reducer plate  94  and straightener  92 . After flow passes through the straightener  92 , it impinges on the lowest baffle  82 . The treatment solution flows upward through each baffle  82  into each successive section  80  and out the top of the column  60 . The column  60  is initially filled with air which is forced out as the surface of the upwardly advancing treatment solution flow passes upward through the column. 
     Now with reference to FIG. 9, a vertical section  80  is enlarged illustrating a plurality of tissue mounts  100  depending from the upper baffle  82 . The tissue mounts  100  comprise U-shaped members  102 , more clearly shown in FIG.  11 . FIG. 10 shows the circumferential array of mounts  100  surrounding the central support rod  84 . Each mount  100  has a generally rectangular configuration and is oriented radially in the baffle  82 . That is, free ends of the U-shaped members  102  insert within similarly sized downwardly opening apertures  104  in the baffle  82 . One of the apertures  104  for each mount  100  is positioned close to the support rod  84 , while the other is positioned close to the tube  61 . The apertures  104  extend approximately halfway through the thickness of the baffle  82  and a smaller diameter through hole  106  continues upward to the top surface of the baffle. This hole  106  is needed to push the mounts  100  from the apertures  104  when treatment is finished. Preferably, the legs of the U-shaped members  102  are spread outward a slight amount so that they have to be squeezed together to fit into the two apertures  104 . This ensures a tight fit so the mounts  100  will not fall out of the apertures  104 . 
     Rectangular tissue pieces  108  are attached to the mounts with sutures or other similar expedient. In the illustrated embodiment, a lower edge  110  of each tissue piece  108  loops around the bridge portion of the U-shaped member  102  and is sewn to the main body of the tissue piece along line  112 . In this way, the leading edge of the tissue piece  108  in the upward flow stream is rounded, and thus protected from friction induced tearing or wear. One or more sutures  114  connect the upper corners of the tissue piece  108  to the upper ends of the legs of the U-shaped members  112 . Preferably the tissue piece  108  is only connected at one or two locations along its vertical length to prevent gross movement or flapping of the tissue, while allowing the maximum freedom for the tissue to shrink. An O-ring  116  or other such device placed on each leg of the member  112  prevents the sutures  114  from sliding down the leg. The upward flow  118  of treatment solution also assists in maintaining the generally planar configuration of each tissue piece  108 . 
     Mounting the tissue pieces  108  in a planar configuration substantially parallel to the direction of flow of the solution ensures that an even amount of solution contacts both sides of the tissue. That is, is the tissue pieces were canted with respect to the flow, the backsides would be exposed to less direct flow, and eddy currents and the like might be set up, further making the fluid exposure nonuniform. In addition, the preferred parallel orientation minimizes any stretching of the tissue during the extensive treatment period, such as might occur if the fluid was directed to one face of the tissue or the other. 
     The radial orientation of the plane of each tissue piece  108  desirably ensures uniform contact with treatment solution during flow through the column  60 . Ideally, the baffles  82  include perforations  120 , seen in FIG. 10, which create the uniform, nonlinear flow. The same velocity of solution is produced at any radial point from the support rod  84  outward. Of course, different pieces of tissue  108  have been shown to possess widely different properties, even from the same pericardial sac. Nevertheless, the present treatment configuration is designed to maximize the uniformity of conditions seen by each piece of tissue  108 . There may be some variation in treatment conditions between the top and bottom reaches of the container due to fluid head differences, but applicants believe that such variations are minimal for the six foot tall column  60  described herein. 
     There are preferably eight vertical sections  80  in which six tissue pieces  108  are mounted for a total of forty-eight tissue pieces being treated at once. Of course, other numbers of sections and tissue pieces per section are possible. The present flow column is extremely well-suited for consistently manufacturing high quality treated bioprosthetic tissue. The segmented flow column with uniform flow, and vertical orientation of each tissue piece  108  provides high uniformity of treatment. The modular nature of the column with the entire support rod  84  having all of the baffles  82  attached thereto is a significant advantage in manufacturing. One batch of tissues may be treated, and then removed so that after flushing the system a new batch can be ready for installation and treatment. Furthermore, the flow column lends itself to a high degree of control over the system parameters such as the relative tissue/fluid velocity and the temperature. Significantly, there are no large stagnant zones of flow within the column, and especially not within each vertical segment  80 . 
     Rat Subcutaneous Studies 
     FIGS. 12 and 13 are results of calcium uptake measurements from tissue treated in a variety of ways, implanted subcutaneously in rats for several months, and then removed. These graphs indicate that heat alone reduces calcium uptake in comparison with a control, and that heat and motion reduces the calcium uptake even further. A number of shaking, stirring or movement apparatuses were used at two different temperatures, with the same general results. 
     FIG. 12 shows the results from three groups of samples of untreated and treated bovine pericardium tissue. The first group (GLUT CONTROL) exhibited an average measurement of about 16% calcium from 12 tissue samples which were subjected to a post-fixation treatment of unheated and static glutaraldehyde. The second group (HEAT) exhibited an average measurement of about 7% calcium from 8 tissue samples which were subjected to a post-fixation treatment of static glutaraldehyde heated to a temperature of 50° C. Finally, the third group (HEAT AND SHAKING) exhibited an average measurement of about 4% calcium from 7 tissue samples which were subjected to a post-fixation treatment of static glutaraldehyde heated to a temperature of 50° C. The treatment solution for all three groups was identical—0.6% HEPES-glutaraldehyde at a pH of 7.4—and the treatment period was equal—2 months. The third group was shaken in a bottle or container using a reciprocal orbital shaker actuated at 80 RPM. The rats were all approximately 12 days old, and the tissue samples were left implanted for eight weeks. 
     FIG. 13 shows the results from a number of groups of samples of untreated and treated bovine pericardium tissue. The calcium uptake results for the groups are indicated by bars with different shading depending on the overall treatment regimen. Thus, the black bars for group  1  are the control (no heat or shaking), the middle shaded bars are for samples subjected to shaking and heat treated to 50° C., and the right-hand white bars are for samples subjected to shaking and heat treated to 42° C. 
     Group  1  on the left is a control and shows results for two subgroups of 7 and 4 samples each. The control samples were treated for 2 months in 0.6% HEPES-glutaraldehyde at a pH of 7.4 with no heat or movement. Each sample was implanted in 16 day old rats, and left implanted for a period of between 3 and 4 months before being removed to test for calcium. 
     Groups  4 - 6  in the middle were all heat treated at 50° C. in the same treatment solution as group  1  for the same period. The differences between the treatment regimen for groups  2 - 6  are the methods used to induce relative tissue/fluid movement. The methods are shown graphically below each group. Group  2 - 7  includes two subgroups of 7 and 8 samples each subjected to reciprocal orbital shaking. Group  3 - 7  includes two subgroups of 2 and 11 samples each placed in a flask with a magnetic stirring bar in the bottom. Group  4  is the same method as group  3  but with two subgroups of 8 samples each placed on a filter instead of being allowed to float around the flask. Group  5  included two subgroups of 12 samples each placed in a first container and subjected to a rolling motion, using a tilted ferris wheel arrangement. Group  6  included two subgroups of 20 and 12 samples each placed in a second container and also subjected to a rolling motion. 
     Groups  7 - 9  on the right were all heat treated at 42° C. in the same treatment solution as groups  1 - 8  and for the same period. Again, the differences between the treatment regimen for groups  7 - 9  are the methods used to induce relative tissue/fluid movement, shown graphically below each group. Group  7  includes two subgroups of 8 and 4 samples each subjected to reciprocal orbital shaking. Group  8  includes two subgroups of 8 and 11 samples each placed in a flask with a magnetic stirring bar in the bottom. Group  9  is the same method as group  8  but with two subgroups of 8 samples each placed on a filter instead of being allowed to float around the flask. 
     It is apparent from these tests that the shaking and heat treatment reduced calcium intake over the control group, as well as over the heat treatment alone. Also, treatment at 50° C. was substantially more effective than treatment at 42° C. Comparisons of the different shaking/stirring methods indicates that stirring with a magnetic rod within the flask produced the least amount of calcium uptake, regardless of temperature, although perhaps not by a significant margin at 50° C. 
     It is understood that the exemplary methods and apparatuses for treating glutaraldehyde fixed biological tissue described herein and shown in the drawings represent only presently preferred embodiments of the present invention. Indeed, various modifications and additions may be made to such embodiments without departing from the spirit and scope of the invention. For example, various fixing agents, such as Denacol® or aldehydes other than glutaraldehyde, may exhibit properties similar to those of glutaraldehyde so as to make them suitable for use in the present invention and, thus, may likewise be utilized. Accordingly, these and other modifications and additions may be obvious to those skilled in the art and may be implemented to adapt the present invention for use in a variety of different applications. Furthermore, the scope of the invention should be determined with reference to the appended claims.