Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
  • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
  • C08H1/00—Macromolecular products derived from proteins
  • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather

Definitions

• the invention concerns the use of a crosslinking agent, such as glutaraldehyde, to process a biological tissue, such as a heart valve.
• a crosslinking agent such as glutaraldehyde
• glutaraldehyde has shown to be beneficial in producing tissues of greater thermal stability, greater flexibility (in comparison to conventional crosslinking techniques) , and increased durability.
• glutaraldehyde is also cytotoxic — even low concentrations of glutaraldehyde require rinsing the tissue to remove residual glutaraldehyde.
• concentrations typically used to crosslink biological tissues about 0.6% v/v, even though as little as 0.2% has been used successfully glutaraldehyde's toxicity is even more of a problem.
• the present invention provides an improved method for crosslinking or fixing collagenous biological tissue, such as a porcine heart valve, using a low concentration of crosslinking agent. The residual levels of crosslinking agent are reduced, while maintaining or enhancing durability and flexibility of the treated tissue.
• the process utilizes an aqueous delivery system, it avoids the use of organic solvents such as acetone, which are not only potentially toxic, but also dehydrate, denature, or destroy collagen-containing substrates (i.e., veins, arteries, heart valves, etc.).
• organic solvents such as acetone
• Other attributes of low-concentration glutaraldehyde fixation include: 1) a relatively higher degree of intramolecular crosslinks provides softer tissue. For heart valves, this means improved hemodynamics; for vascular grafts, this means greater kink resistance. 2) A relatively higher degree of short chain intramolecular crosslinks (along the collagen strand) , which may also provide the tissue with protection from sources known to cause chain scission reactions in the collagen protein matrix.
• While the present invention is suitable for a number of vascular prostheses, it is of particular value in the field of small diameter vessel replacement. It is useful in coronary access bypass procedures, particularly when the patient has had previous coronary replacement surgery and adequate saphenous veins or internal mammary arteries no longer exist due to previous excision. Since the prosthesis made in accordance with this invention may have patency equivalent to or better than that of a saphenous graft, the use of such a prosthesis would alleviate the need for saphenous vein excision. It is also applicable to systemic microvascular vessel replacement, such as in the hand or foot.
• FIGURES Figure 1 compares tensile strength values of bovine carotid arteries crosslinked in 0.01% glutaraldehyde vs. 0.05% glutaraldehyde.
• Figure 2 compares suture retention values of bovine carotid arteries crosslinked in 0.01% glutaraldehyde vs. 0.05% glutaraldehyde.
• Figure 3 shows the effect of different fixation conditions on tensile and suture retention strengths of bovine median arteries.
• Figure 4 shows the effect of different fixation conditions on bursting strength of bovine median arteries.
• FIGS 5a and 5b illustrate the difference between low concentration glutaraldehyde intramolecular
• Figure 6 compares the leaflet shrink temperature of leaflets crosslinked in 0.5% glutaraldehyde vs 0.05% glutaraldehyde.
• Figure 7 shows the effects of e-beam radiation on pressure drop.
• the fixing solution is a buffered solution containing glutaraldehyde.
• biological tissue refers to a collagen-containing material which may be derived from different animal species, typically mammalian.
• the biological tissue is typically a soft tissue suitable for implantation, such as bioprosthetic tissue or the like, but the invention should not be limited thereby.
• Specific examples include, but are not limited to, heart valves, particularly porcine heart valves; * aortic roots, walls, and/or leaflets; pericardium, preferably bovine pericardium or the like, and products derived from pericardium, such as a pericardial patch; connective tissue derived materials such as dura mater; homograft tissues, such as aortic homografts and saphenous bypass grafts; tendons.
• RECTIFIED SHEET(RULE91) ISA/EP ligaments, skin patches; blood vessels, particularly bovine arteries and veins, and human umbilical tissue, such as veins; bone; and the like. Any other biologically-derived materials which are known, or become known, as being suitable for processing in accordance with the invention are within the contemplation of the invention.
• the biological tissue explanted from its source, may be processed in any suitable manner prior to exposure to a crosslinking agent.
• the biological tissue is carefully cleaned and then treated with a filtered proteolytic enzyme concentrate to digest and thereby substantially eliminate antigenic tissue from the biological tissue.
• the cleaning step typically involves stripping the adventitia and undesirable fat and muscle tissue from the tissue.
• Exposing the biological tissue to a crosslinking reagent refers to any method of contacting the biological tissue with the crosslinking agent.
• exposing the tissue to a crosslinking agent refers to immersing the tissue in the crosslinking agent.
• a crosslinking agent such as an aldehyde, e.g., glutaraldehyde
• glutaraldehyde e.g., glutaraldehyde
• monomeric glutaraldehyde is deposited directly to the surface of the substrate. Regardless of the initial concentration of the solution prior to vaporization, pure glutaraldehyde is in contact with the tissue. The concentration in the tissue is therefore very difficult, if not impossible, to control.
• the glutaraldehyde that is typically used in accordance with the invention is a biological grade 50% solution commercially available, from, for example. Electron Microscopy Sciences (Fort Washington, PA) . Such commercially available glutaraldehyde may also be available in a variety of other grades, purities, and/or concentrations. A biological grade of glutaraldehyde typically does not require additional purification, but it may be desirable to pass the final crosslinking solution through a 0.2 ⁇ pore size hydrophilic membrane filter to remove any biological contaminants.
• a biological tissue may be exposed to a fixing solution comprising glutaraldehyde in a suitable buffer.
• suitable buffers for use in the practice of the invention are those buffers which have a buffering capacity sufficient to maintain a physiologically acceptable pH, do not cause substantial deleterious harm to the biological tissue, and/or do not interfere with the treatment process.
• Exemplary buffers include, but are not limited to phosphate-buffered saline (PBS) , and organic buffers, such as N-(2-hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES) or morpholine propanesulphonic acid (MOPS) ; and buffers which include borate, bicarbonate, carbonate, cacodylate, or citrate.
• PBS phosphate-buffered saline
• organic buffers such as N-(2-hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES) or morpholine propanesulphonic acid (MOPS)
• buffers which include borate, bicarbonate, carbonate, cacodylate, or citrate.
• the preferred fixing solution is less than 0.1% v/v glutaraldehyde in a citrate buffer.
• parameters in the treatment protocol may be varied according to achieve a particular purpose. These parameters include, but are not limited to glutaraldehyde concentration, solution composition, pH and ionic strength, time and temperature of biological tissue exposure to glutaraldehyde, and the ratio of tissue to volume of solution, and the biological tissue configuration during the initial fixation.
• the invention is not to be limited thereby.
• the crosslinked biological tissue is then rinsed, using, for example, any suitable rinsing or laving material.
• the rinsing agent is sterile, physiological saline.
• the invention may include additional processing.
• the crosslinked biological tissue may be exposed to one or more anticalcification agents, one or more bioburden reduction agents, at least one rinsing solution, and one or more sterilizing agents or protocols.
• the biological tissue, crosslinked in accordance with the invention may be stored for up to about one year or more prior to final sterilization. Exemplary additional processing is described in more detail below. BIOBURDEN REDUCTION
• An embodiment of the invention may include exposing the crosslinked biological tissue to one or more bioburden reduction agents, typically for up to about 10 hours, preferably for about 2 to about 4 hours.
• a porcine heart valve treated with glutaraldehyde as noted above may then be exposed to a buffered solution containing about 1-5% glutaraldehyde, about 1-6% formaldehyde, and about 15-25% ethanol.
• Typical buffers include PBS, HEPES, phosphate, and citrate buffers.
• An embodiment of the invention may include storing the crosslinked biological tissue for up to a year or more prior to final sterilization.
• a porcine heart valve crosslinked with glutaraldehyde in accordance with the invention may be temporarily stored at room temperature in a 0.5% glutaraldehyde solution buffered with PBS, citrate or the like.
• ANTICALCIFICATION TREATMENT An embodiment of the invention may include exposing the crosslinked tissue to one or more reagents designed to reduce or inhibit calcification of the biological tissue after implantation. A number of anti- calcification reagents are known in the art.
• the crosslinked biological tissue may be exposed to an alcohol and/or an aluminum salt in order to reduce or inhibit calcification.
• the crosslinked biological tissue may be immersed in a solution containing greater than about 50% of a lower aliphatic alcohol for a period sufficient to render the biological tissue resistant to calcification, typically up to about 96 hours.
• the alcohol is preferably a lower aliphatic alcohol (Cl to C3) , such as methanol, ethanol, propanol or isopropanol. In a preferred embodiment, the alcohol is ethanol.
• an illustrative exposure time is preferably between about 24 to 96 hours.
• the anticalcification agent may include a multivalent metallic cation, such as a salt of aluminum or iron.
• a multivalent metallic cation such as a salt of aluminum or iron.
• the crosslinked biological tissue may be immersed in a solution containing from about 0.1M to about 0.001M A1C1 3 for a period sufficient to render the biological tissue resistant to calcification.
• the crosslinked biological tissue may be stored in an alcohol-glutaraldehyde solution, preferably in an amount sufficient to maintain calcification inhibition and/or sterility.
• the biological tissue may be stored in a buffered alcohol solution containing glutaraldehyde, typically greater than about 60%, and preferably between about 60% and about 80%, alcohol and less than about 0.5%, preferably between about 0.2% to 0.5%, glutaraldehyde.
• the biological tissue, calcification-inhibited and/or crosslinked, may then be placed or packaged in a container.
• the biological tissue is packaged and sealed, in physiological saline, in its final container prior to terminal sterilization.
• Packaging preferably means placing in a container suitable for sterilization, storage, and/or shipping.
• the container may be constructed of glass or polymeric plastic, such as polypropylene, polyethylene, and/or epoxies. It is intended that the invention should not be limited by the type of container and seal being employed; other materials may be used, as well as mixtures, blends, and/or copolymers.
• the crosslinked, packaged biological tissue may then be sterilized, as noted below, or it may be stored for up to about a year or more prior to sterilization.
• storage includes long term storage, e.g., six months, twelve months, or for up to about five years or more.
• a method in accordance with the invention may also include sterilizing the tissue.
• sterilizing refers to exposing the biological tissue to a sterilizing beam of accelerated electrons, i.e., an electron beam (e-beam).
• the particle beam which comprises the e-beam preferably includes directional bombardment, i.e. , bombardment from one direction only, and includes single-side or multiple-side irradiation.
• the biological tissue, crosslinked in accordance with the invention may be sterilized, preferably after the biological tissue has been packaged. Suitable sterilizing protocols include, but are not limited to x-ray or gamma radiation, e-beam radiation, and the like.
• the preferred method of sterilizing the crosslinked tissue is by exposing the biological tissue, packaged in saline, to accelerated electrons.
• the biological tissue may be subjected to the electron beam until a dose of approximately 25 kilogray (kGy) is achieved, or approximately 1-10 minutes, depending on the dimensions of the material.
• kGy kilogray
• a major advantage of e-beam processing over conventional gamma radiation is the processing speed or the high rate at which the energy can be applied in a controlled manner, which usually translates to lower sterilization costs.
• biological tissues are treated by exposing the tissue to e-beam radiation sufficient to effect sterilization.
• the present invention provides a biological tissue sterilized by e-beam radiation, with the resulting biological tissue exhibiting enhanced performance 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 handling protocols. Further, the methods and tissues of the present invention, which use 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 chemicals. Moreover, the amount of radiation required for e-beam sterilization does not significantly degrade the biological tissue, thus providing a more durable transplantable tissue.
• the dose rate for gamma radiation is approximately 110 grays per minute and the dose rate of e-beam is approximately 7800 grays per minute. Consequently, exposure times are dramatically greater for gamma radiation, which requires low doses over an extended period to effect sterilization.
• the high dose rates involved in e-beam irradiation promote diffusion of oxygen into biological tissue at a rate insufficient to participate in free radical formation reactions, such as 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 container may be minimized.
• the high dose rate of e-beams relative to gamma rays permits a higher processing rate of sterilization, commonly an order of magnitude higher.
• gamma radiation penetrates approximately ten times further into materials than 10 MeV electrons in the same material.
• Electron beams provide high-energy electrons to the exterior of the material, which penetrate the material, and in turn put subsequent electrons in motion.
• oxygen is not capable of diffusing into the material at a rate required to participate in oxidative reactions that may lead to degradation of the material.
• 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.
• the amount of e-beam radiation is an amount sufficient to sterilize the biological tissue, and in some embodiments, an amount sufficient to sterilize the biological tissue packaged in its final container.
• One skilled in the 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.
• 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) .
• Kgy kilograys
• One kilogray is equal to one thousand joules of energy deposited per kilogram of material.
• the biological tissue may be irradiated until a dose of approximately 25 Kgy or more is achieved.
• Effective sterilization may be easily determined using conventional microbiological techniques, such as for example, the inclusion of suitable biological indicators in the radiation batch or contacting the tissue with a culture medium and incubating the medium to determine sterility of the tissue. Dose may also be determined with the use of radiochromic dye films. Such films are calibrated, usually in a gamma field, by reference to a national standard.
• Degradation of the biological tissue by 5 irradiation may also be determined using well known and conventional 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
• 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
• the biological tissue may be exposed to a buffered glutaraldehyde solution from about 4°C to about 37°C, preferably at about 20°C; at a pH from about 6 to about 8, preferably 6.3 to 6.5; and for a period up to about 10 days,
• the aforementioned steps, in combination, produce a prosthesis having greater strength and pliability, reduced antigenicity, and greater ease of use than prostheses produced using other processes.
• the biological tissue, crosslinked in accordance with the invention may be packaged in a solution of approximately 0.5% glutaraldehyde.
• the biological tissue may be thoroughly rinsed with sterile, physiological saline (e.g., 0.9% sodium chloride) .
• physiological saline e.g. 0.9% sodium chloride
• the purpose of the saline rinse is to reduce the concentration of residual glutaraldehyde present on the tissue surface and in the interstitial spaces of the tissue, thus minimizing the chance of a toxic response in the patient.
• the biological tissue may be placed into a plastic or polymeric container, filled with sterile saline, and capped or sealed. The capping of the product should be a permanent seal that should not be opened until the time of the implant.
• the present invention provides an improved biological tissue in view of its strength, pliability, and reduced antigenicity. These characteristics are particularly desirable in view of the stressful procedure required to implant the biological tissue and the possibility of rejection by the body.
• the biological tissue With respect to the implantation procedure for some biological tissues, the biological tissue must have resistance to kinking, good suture puncturability, and the ability to readily seal suture holes. Suture retention, i.e., the ability to resist tensile force, must be high. Additionally, the graft must have high resistance to bursting to withstand possible high systolic pressures and to guard against aneurysm formation. Grafts that are insufficiently strong and/or are too antigenic may be potentially fatal to the patient.
• the prostheses produced according to the present invention may be packaged as a kit, preferably sterile wet-packaged in a 0.9% NaCl solution.
• the selection of such additional elements are well within the ordinary skill in the art.
• EXAMPLES Example 1. 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 overnight. Extraneous tissue such as adipose, skeletal muscle, myocardium, bone, trachea, etc.
• a 50 mM citrate buffer solution is prepared per the following formula (10 liters) : To 9.0 liters of sterile, de-ionized water, add:
• the exposure of tissue to the glutaraldehyde solution proceeds for a period of time ranging from about 24 to about 120 hours, depending on the concentration of glutaraldehyde in the solution.
• a high glutaraldehyde concentration corresponds to a short fixation time; a low glutaraldehyde concentration corresponds to a long fixation time.
• an exposure time of approximately 72 hours is sufficient to maximize the crosslink density within the interstices of the tissue. This corresponds to a shrink temperature of approximately 80-89°C, depending on the type of tissue used.
• the tissue is submersed in a sterilant solution containing 2% (v/v) glutaraldehyde, 3% (v/v) formaldehyde, and 20% (v/v) ethyl alcohol.
• This multi-component sterilant reduces any residual bioburden on the tissue prior to rinsing and packaging.
• tissue is then thoroughly rinsed with sufficient sterile saline to minimize the presence of the processing chemicals. This typically requires applying four or five 10 liter aliquots over a 24-hour period. The exposure time should be watched carefully, since diffusion of residuals from the tissue is a time- dependent phenomenon.
• tissue is placed in a sterile container (valve jar, vascular graft vial, etc.) and filled with sterile saline. The package is then permanently sealed. Note: 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-beam sterilization.
• Example 2 Two identical experiments were performed to evaluate the effects of different concentrations of glutaraldehyde on the physical properties of bovine carotid artery vascular protheses. Arteries were received in cold, sterile saline (0.9% sodium chloride) . Extraneous tissue such as adipose, bone, cartilage, connective tissue, etc., was stripped from the vascular tissue. The arteries were enzymatically digested with a citrate-buffered ficin solution to remove a specified portion of smooth muscle tissue. The arteries were thoroughly rinsed and placed into one of two tanks containing glutaraldehyde to crosslink the collagen component of the tissue.
• Extraneous tissue such as adipose, bone, cartilage, connective tissue, etc.
• One tank contained 50mM citrate-buffered 0.01% glutaraldehyde, the other tank contained 50mM citrate-buffered 0.05% glutaraldehyde.
• the arteries in the 0.01% solution were allowed to crosslink for approximately five days and the arteries in the 0.05% solution were crosslinked for approximately two days. Upon completion of the crosslinking reaction. all arteries were placed in a 50mM citrate-buffered 2% glutaraldehyde solution as a sterilization step.
• the Radial Tensile Strength test involves cutting a piece of crosslinked carotid artery, making a longitudinal incision, and pulling the tissue in a radial orientation until failure on an apparatus such as an Instron.
• the Suture Retention Strength test involves inserting a loop of surgical suture, such as a 5-0 PTFE-impregnated polyester suture, through a tissue sample a specified distance from a cross-sectionally cut edge. This distance, the bite size, may be, for example, about 3 mm. The suture is pulled until the tissue fails on an apparatus such as an Instron.
• the results of the Tensile Strength and Suture Retention Strength testing is displayed on Figure 1 and 2, respectively.
• Retention Strength specifically, strength for both concentrations is lower in Batch 2 and Batch 1. This is most likely a result of a difference in the enzymatic digestion processes. Each ficin solution varies slightly in enzymatic activity. In other words, it is possible that the digestion solution prepared for Batch 2 had a slightly higher enzyme activity than that for Batch 1. Nevertheless, the differences in strengths within the batch are negligible.
• Example 3 An experiment was performed to evaluate the effects of different concentrations of glutaraldehyde on some of the physical properties of bovine median artery vascular protheses. Processing of tissue was the same as that described in Example 2 until the crosslinking step. The digested arteries were placed in one of three tanks for crosslinking.
• Tank 1 contained 50mM citrate- buffered 0.01% glutaraldehyde
• tank 2 contained 50mM citrate-buffered 0.075% glutaraldehyde
• tank 3 contained 50mM citrate-buffered 2.0% glutaraldehyde.
• the arteries were allowed to remain in each of the fixation tanks until the crosslinking reaction was completed, or about four days. Upon completion of the crosslinking reaction, half the arteries from tanks 1 and 2 were placed in a 50mM citrate-buffered 2% glutaraldehyde solution as a sterilization step. The other half remained in the original solution. After 24 hours, all arteries were placed in individual glass vials containing 40% ethyl alcohol, and capped. Samples from each group were then subjected to the following test: Radial Tensile Strength, Suture Retention Strength, and Bursting Strength.
• Figure 3 shows that the radial tensile strength and suture retention strength are very similar for tissue processed at each of the conditions in this experiment.
• the extremes 0.01% and 2.0%) are of particular interest because this data represents the lower end of the range.
• This data contradicts the very common notion in the industry that maintains that high concentration fixation leads to high thermal stability, which leads to high strength.
• These results, as well as the shrink temperature data in Figure 6 show that this is not the case.
• the fact that the 0.01%/2% group has a sightly lower tensile strength than the other groups is considered an artifact. It is expected that the subsequent treatment with 2% glutaraldehyde will, if anything, strengthen the tissue, not weaken it.
• Figure 4 shows the results of the Bursting Strength testing, where a vascular prosthesis sample is inflated with water until it bursts. The internal pressure at the time of failure is recorded as the bursting strength. Again, looking at the extremes, results are nearly identical, even though the low- glutaraldehyde test group was fixed at a glutaraldehyde concentration l/200th of conventional 2% crosslinking.
• Figures 5a and 5b are simplified representations of the formation of a short-chain intramolecular crosslinksproducedby low-glutaraldehyde and long-chain intermolecular crosslinks produced by high-glutaraldehyde fixation, respectively. These Figures aid in showing that, in the presence of intramolecular crosslinks, the collagen strand may maintain much of its integrity, even if peptide bonds are cleaved by radiation exposure.
• Tissue crosslinked with low glutaraldehyde forms a higher density of intramolecular crosslinks, and this may be expressed in terms of a higher shrink temperature than conventionally-fixed tissue.
• This relationship is shown by crosslinking two groups of fifteen porcine aortic valve leaflets with 0.5% or 0.05% glutaraldehyde. Shrink temperatures were measured via Differential Scanning Calorimetry and the results are contained in Figure 6.
• Figure 8 compares the 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 radiation.
• Effe ⁇ tive 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.
• EA Effective Orifice Area
• ⁇ P mean positive pressure drop, in mm Hg
• the theory behind enhanced hemodynamics in irradiated tissue heart valves involves the disruption of molecular bonds which hold the collagen triple helix intact.
• the intramolecular crosslinks offered by this technology serve as reenforcement to the collagen backbone as its own structural frame work is weakened by the radiation.
• Example 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 to withstand 200 million cardiac cycles on an accelerated wear tester. This translates to approximately five years of real time. At some point in the future, 380 million cycles of the same testing may be required.
• Group 1 Crosslinked in 0.03% glutaraldehyde; stored in 0.5% glutaraldehyde (e-beam negative control) .
• Group 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; rinsed for removal of residuals; stored in 0.9% sodium chloride; e-beam sterilized, 25kGy.
• Group 4 Crosslinked in 0.5% glutaraldehyde; rinsed for removal of residuals; stored in 0.9% sodium chloride; e-beam sterilized, 25 kGy (concentration negative control) .
• Results of this experiment are located in Table 1 below. These results clearly indicate that, compared to control valves (Groups 1 and 4) , exposing the tissue valves to e-beam radiation does not have a negative effect on durability after in vitro 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 for anticalcification.
• Group 4 3 3 holes (0.5 to 3 mm)
• Example 7 A vascular graft prosthesis, for example an artery, is stripped of its adventitia, fat and muscle tissue, and digested in an activated protease. This stripped and cleaned artery is treated with succinic anhydride at a controlled basic pH to produce a negative charge on the surface of the artery. The negatively charged artery may then be treated with glutaraldehyde in a concentration ranging from about 0.005 to about 5% in a citrate buffer to fix and strengthen the artery by cross-linkage. The preferred concentration is less than about .01%.
• low concentrations of glutaraldehyde typically yield softer and more flexible products, based on qualitative comparative assessments of graft products cross-linked using low and high glutaraldehyde concentrations.
• Qualitatively, increased pliability of graft products crosslinked using low glutaraldehyde concentrations was demonstrated by assessing the radius of curvature of the graft; the radius is smaller in low glutaraldehyde concentration fixed product than in a high glutaraldehyde concentration fixed product.
• the fixed graft may then be sterilized using glutaraldehyde at a concentration range of about 1% to about 4%, preferably about 2%.
• This combination of steps produces a prosthesis having increased strength, increased durability, increased pliability, and decreased thrombogenicity.
• the digestion step in the present invention comprises using an activated protease to remove antigenic material from the surface of the graft. While it is known to utilize the proteolytic enzyme ficin as part of the cleaning step in preparing a blood vessel, the present method provides more effective elimination of antigenic tissue from the natural graft prosthesis through the use of an activated protease. What remains after the digestion step is a collagen matrix.
• Example 8 After harvesting, the graft is digested in a protease, preferably, an activated protease, to remove antigenic substances from the graft.
• the activated protease is ficin, more preferably, filtered ficin that is activated by adding cysteine to the ficin concentrate.
• a buffer is used comprising citric acid and sodium citrate, although other buffers may be used. The specific amount of ficin used in the practice of this invention depends on the activity of the ficin used, since variances in the activity level of enzymes are normal.
• the exact amount of ficin required to manufacture grafts is typically adjusted each time grafts are manufactured to achieve a constant volumetric activity; i.e., the determination of the specific quantities of ficin to be used during graft production typically requires that the ficin be evaluated for activity and that adjustments to the amount of ficin used be made in accordance to the activity noted.
• the concentration of ficin typically used according to the present invention amounts to the addition of approximately 65 grams of ficin to a reactor volume of about 20 liters.
• the ficin activity of the digesting solution is about 9.8 mmol NPZG/liter-minute.
• the temperature and pH should be monitored. In one embodiment, the temperature should be in the range between 30°C and 50°C, more preferably 40°C + 2°C. The pH should be in the range between 5 and 7, more preferably 6.3 + 0.1.
• the digestion time utilized is not critical, but should be sufficient to remove any antigenic material; 2-3 hours is typically sufficient.
• the graft should then be rinsed several times in distilled water. In a preferred embodiment, the graft is rinsed 4 times. Digestion is terminated in a stop bath, preferably a stop bath containing sodium chlorite. In a preferred embodiment. the concentration of sodium chlorite is 0.1%. The graft is then rinsed again, preferably 4 times.
• Example 9 Using a hemostat, suspend one artery from a ring stand. Using a clean scissors, strip adventitia, fat, and muscle tissue from the artery. Place the artery in a clean beaker with clean 0.9% sodium chloride on ice. Repeat above steps until all arteries have been stripped.
• Tie off side branches with a suture Tie off as close to the main artery as possible with a triple surgeon's knot. Trim excess side branches near the knot. Remove the artery from the ring stand when all side branches have been tied.
• the concentrated ficin solution is made to the equivalent of 85 grams/liter of Sigma latex powder. Place 2.8 liters of ficin buffer in a flask in the 40°C water shaker bath. Approximately 65 grams of ficin powder are required. The ficin powder is dissolved in the 40°C buffer and filtered through a 5 micron Gelman Aero 50A filter.
• For the stop bath dissolve 20 grams of sodium chlorite into 20 liters of H 2 0. Shut off the pumps and stirrer. Open drain valve and allow the tank to empty. Close the drain valve and fill the reactor with 20 liters of H 2 0. Turn on the pumps and stirrer and allow to equilibrate for several minutes. Repeat for a total of four rinses. Shut off the pumps and stirrer and open the drain valve to allow the tank to empty. Close the drain valve and fill the reactor with the stop bath solution. Start the pumps and stirrer. Run the stop bath for 15- 20 minutes to inactivate any residual enzyme. Drain the reactor and perform four full rinses.
• Biological tissues can be produced as follows: 1. individual tissue valves are exposed to
• the crosslinked biological tissue can then be initially sterilized in a multi-component sterilant of 2% glutaraldehyde, 3% formaldehyde, and 20% ethanol in an aqueous buffer.
• the initially sterilized biological tissue may then be subjected to ethanol extraction (60% ethanol), optionally including 0.1M A1C1 3 , and a 24 hour wash in saline.
• the biological tissue is then packaged in a saline solution and subjected to terminal sterilization by exposing the tissue to e-beam radiation.

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• 1995
  • 1995-06-15 JP JP8502508A patent/JPH09502379A/ja active Pending
  • 1995-06-15 WO PCT/US1995/007679 patent/WO1995034332A1/en not_active Application Discontinuation
  • 1995-06-15 BR BR9505496A patent/BR9505496A/pt not_active Application Discontinuation
  • 1995-06-15 EP EP95923929A patent/EP0713400A1/de not_active Withdrawn
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  • 1995-06-15 AU AU28634/95A patent/AU2863495A/en not_active Abandoned
  • 1995-06-15 CN CN95190727A patent/CN1131913A/zh active Pending
• 1996
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