PROCESS FOR DECREASING RESIDUAL ALDEHYDE LEVELS IN IMPLANTABLE BIOPROSTHETIC TISSUE

Abstract: Residual aldehyde levels in bioprosthetic tissue are reduced by contacting the tissue with a rinsing solution such that aldehyde which has diffused out of the tissue into the rinsing solution comes into contact with, and reacts with, an amine, thus causing further diffusion of aldehyde out of said tissue. The amine may be dissolved in said rinsing solution or may be immobilized on a solid support to which the rinsing solution is periodically or continuously exposed.

Description: The present invention relates to a method for preparing biological tissue for surgical implantation in bioprosthetic applications. More specifically, the present invention provides a method for reducing residual aldehyde levels in bioprosthetic tissue which has been contacted with an aldehyde during pre-implantation processing. Surgical implantation of biological tissue is well known, and has many clinical applications. The source of the tissue may be the patient himself (autologous tissue), a member of the same species as the patient (homologous tissue), or a member of a different species (heterologous tissue). Examples of tissues which have been implanted as bioprosthetic materials are tendons, ligaments, heart valves, bone, skin patches, collagen, pericardial tissue patches, arteries, and many others. The introduction of methods for rendering tissues nonantigenic has made it possible to transplant tissue derived from various animal species into humans to repair or replace defective body parts. The preparation of biological tissue for implantation frequently includes treatment with a chemical agent that "fixes" (i.e., "tans") the tissue. Various aldehydes have been used for this purpose, including glyoxal, formaldehyde, dialdehyde starch, and glutaraldehyde. Glutaraldehyde is considered by many to be the preferred tanning agent, and is also used as a tissue sterilant, having proven to be a good barrier against microbial contamination. Glutaraldehyde treatment is also known to render tissue substantially non-antigenic so that transplanted heterologous tissue does not elicit an adverse immune response in the recipient. Aldehyde-treated tissue is commonly rinsed with sterile distilled water or a solution such as saline prior to surgical implantation, in an attempt to remove residual aldehyde compounds from the tissue. Certain post-implantation problems, such as inflammation and other adverse reactions in some patients, are believed in some cases to be caused by residual aldehyde in implanted bioprosthetic tissue. Accordingly, it is the object of the present invention to provide a method for reducing said residual aldehyde in tissue to be implanted without altering the physical properties of the tissue and while maintaining aseptic working conditions. EP-A-0 174 737 discloses a process for the treatment of collagenous tissue in order to adapt it for use in a prosthetic implant and to promote the growth of endothelial cells thereon after implantation. This process includes treating a glutaraldehyde-fixed tissue with an agent which inhibits calcification for a time sufficient to bond substantially all the free reactive groups of the bonded glutaraldehyde molecules to the reactive amino groups of the calcification - inhibiting agent which is an amino diphosphonate. Similarly FR-A- 2 306 707 discloses a process for decreasing the antigenicity of tissue suitable for implantation which involves mixing and reacting the tissue following the glutaraldehyde treatment with an amine or amino acid capable of blocking all free functional groups of the bound glutaraldehyde. FR-A- 2 298 312 relates to a process for preparing a flexible segment of umbilical cord vessel for use in a prothesis element, comprising rinsing the glutaraldehyde treated segment with a saline solution of a dilute solution of sodiumbicarbonate and then reacting the residual glutaraldehyde with an amino acid to separate it out completely. The object of the present invention is achieved by a method for decreasing aldehyde levels in bioprosthetic tissue previously contacted with said aldehyde to one part per million (1 ppm) or less, comprising contacting said tissue with a rinsing solution, contacting and reacting the aldehyde which has diffused out of said tissue into said solution with an amine which is bound to a non-toxic solid support and which is contained in said rinsing solution in a concentration of from 0.001M to 1.0M, whereby the rinsing solution is maintained at a pH of between 7.0 and 7.6 and at a temperature of between 4°C and 45°C. Binding of the aldehyde by the amine in the rinsing solution maintains an equilibrium favoring diffusion of additional unbound aldehyde out of the tissue. When employing the present invention the residual aldehyde levels in bioprosthetic tissue are reduced prior to surgical implantation of the tissue. When the tissue (which previously has been treated with an aldehyde) reacts with a solution comprising an amine compound in accordance with the present invention which will react with the aldehyde compound the biological tissue will not be harmed. Many types of compounds which are reactive with aldehydes are known to one skilled in the art (see, for example, Morrison and Boyd, Organic Chemistry , 3rd Ed. , Allyn and Bacon, Inc., Boston, 1973, pp. 630-634, herein incorporated by reference). The tissue is contacted with a solution comprising an amine such that the unbound aldehyde which diffuses out of the tissue reacts with the amine. Binding of the aldehyde in the rinsing solution by the amine (through chemical reaction) is thought to create a concentration gradient which promotes further diffusion of aldehyde out of the tissue. The amount of residual unbound aldehyde which diffuses out of the tissue is thus greater than the amount that would diffuse out of the tissue if the rinse solution lacked a compound which reacts with the aldehyde. Using the method of the present invention, the residual glutaraldehyde levels and cytotoxicity of glutaraldehyde-treated tissues have been greatly diminished when compared to glutaraldehyde-treated tissues rinsed conventionally in saline for similar periods of time. The biological tissue which is to be implanted may be derived from many different animal species. Suitable sources include for example bovine, porcine, equine, ovine, kangaroo, rabbit, or human tissues. The tissue may be epithelial or fibrous connective tissue, such as pericardial tissue, dura mater , fascia lata , amnion, tendon, ligament, cartilage, and the like. Alternatively, collagen (including reconstituted collagen and collagen when backed by synthetic fabric), may be used. Many other tissue types, including arteries, veins, skin patches, and bone, are also known to have bioprosthetic uses. The tissue is extracted from its source and processed by one of many known procedures used to prepare biological tissue for implantation as bioprosthetic material. These tissue handling conditions are conventional, and are not considered to be part of the present invention. In general, the tissue is extracted from the animal source, and extraneous material (e.g., fat) is removed. The tissue is then transported, stored, and processed under physiologically acceptable conditions of temperature and pH, i.e., conditions that are not deleterious to the tissue components. The pH range is from 7.0 to 7.6, with a pH of 7.1 to 7.4 being most preferred. Buffers which contact the tissue should not interfere with any of the processing steps. Such buffers have a buffering capacity sufficient to maintain a physiologically acceptable pH, particularly during the fixation of the tissue. The choice of the appropriate buffer and its concentration will depend upon specific tissue preparation conditions, and variations of these buffers have been introduced by several manufacturers. The buffer can be either conventional 0.01-0.02M phosphate-buffered saline (PBS) or phosphate-deficient solutions such as those containing less phosphate than these 0.01 to 0.02M PBS solutions, and preferably less than about 0.001 to about 0.002M phosphate. Preferred buffers in accordance with the present invention include borate, carbonate, bicarbonate, cacodylate (found to be non-toxic in animals), and other synthetic, artificial, or organic buffers such as N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES); morpholine propanesulphonic acid (MOPS); and 1,4- piperazinediethanesulphonic acid (PIPES). Preferably, the buffered or unbuffered solutions used in accordance with the present invention should not interfere with the tissue-stabilizing process afforded by the fixing agents such as glutaraldehyde. That is, they should not react with the fixing agent or prevent the fixing agent from achieving proper fixation of the tissue. Illustrative of these unsuitable buffers are those containing primary and secondary amines such as tris(hydroxymethyl)aminomethane (tris), which react with the aldehyde groups of glutaraldehyde and thus interfere with the normal tissue stabilization process. Treatment of extracted biological tissue with aldehyde compounds (e.g., to fix and/or sterilize the tissue) is well-known in the art. Procedures for such treatment are given, for example, in U.S. -A- 4,120,649; 3,988,782; and 4,553,974, each of which is incorporated herein by reference. Glutaraldehyde is the preferred fixing agent and the tissue advantageously is contacted with a buffered solution containing from about 0.2 to about 0.8 percent (w/v), preferably about 0.6 percent (w/v) glutaraldehyde. The tissue advantageously is contacted with the glutaraldehyde for at least a time sufficient to cross-link proteins in the tissue to a degree sufficient to stabilize the tissue and to render it substantially non-antigenic. The processing procedures may vary according to the type of tissue being processed and its intended application. For example, soft biological tissues may be chemically treated according to any of several known procedures to mitigate post-implantation calcification of the bioprosthetic tissue. Such calcification mitigation treatments are described in, for example, U.S. -A- 4,323,358 and 4,481,009. The tissue can be sterilized by any conventional means, including exposure to ethylene oxide or immersion in a solution containing glutaraldehyde or formaldehyde. A sterilizing solution containing from about 0.2 to about 1% glutaraldehyde or about 4-5% formaldehyde may contain additional substances such as ethanol, surfactants, and buffering compounds. Alternatively, tissue can be sterilized by exposure of frozen or freeze-dried tissue samples to gamma-irradiation. The order of the processing steps may vary. Once the sterilization step has been performed, the tissue must, of course, be maintained under sterile conditions and contacted only with sterile solutions and reagents. After the tissue has been treated according to the method of the invention to reduce residual aldehyde levels, the tissue should not, of course, be contacted with any aldehyde compound in a subsequent step. For example, the tissue should not be treated with a sterilizing solution containing glutaraldehyde or formaldehyde after the treatment to reduce residual aldehyde levels has been completed. Tissue which has been contacted with an aldehyde during pre-implantation processing is then treated by the method of the present invention to reduce residual aldehyde levels in the tissue prior to surgical implantation. A high residual level of glutaraldehyde in bioprosthetic tissue is though to be a cause of certain adverse host reactions, such as inflammation in surrounding host tissues, after implantation of bioprostheses. Indeed, glutaraldehyde-tanned bioprosthetic tissues have shown high cytotoxicity (as measured by in vitro assays) when post-tanning rinses were conventional rinses in saline solution, as often practiced in the art. Removal of residual glutaraldehyde is particularly difficult for thick and/or dense biological tissues, such as ligaments. Using the method of the present invention, residual glutaraldehyde levels have been substantially reduced in bioprosthetic tissues, including ligaments, as described in the "Examples" below. Reduction of residual aldehyde to acceptable levels is accomplished in a relatively short time. In accordance with the method of the present invention, aldehyde-treated biological tissue is contacted with a rinsing solution containing an amine compound which is bound to a non-toxic solid support. Such amine compounds include compounds which contain one or more aldehyde-reactive amino groups. The amine compound may be a primary or secondary amine. Advantageously, amines which react efficiently with aldehyde compounds are used in the method of the invention. Primary amines are generally more reactive with aldehydes than are secondary amines, and primary amines therefore are preferred. Primary amines have the general formula R-NH₂, where R can be an aliphatic (straight-chain or branched) or aromatic group, or a combination thereof. Examples of aliphatic primary amines are methylamine, ethylenediamine, tert -butylamine, ethanolamine, hydroxylamine, and many others known to those skilled in the art. Examples of aromatic primary amines are phenylhydrazine, aniline, toluidine, anisidine, and many others. The rate of reaction of the amine with the aldehyde groups is influenced by the bulk of the R group and by the solubility of the amine compound. In general, the more soluble the amine, and the smaller the R group, the more reactive the compound is, and, therefore, the more preferable the amine compound is for use in the invention. Amines having R groups which are electron-donating, as opposed to electron-withdrawing, are also preferred, because they are more basic, and therefore more reactive in nucleophilic additions to a carbonyl group of an aldehyde. Since many amines are known to be toxic, the toxicity of a particular amine, and of the product of its reaction with an aldehyde, should be considered. However, since the amine is bound to a solid support, in accordance with the invention, relatively toxic amines can be used as long as the bond to the solid support is stable and non-reversible so that the amine compounds could not become free in the solution. Primary amines which are non-toxic and therefore preferred for use in the method of the invention include the amino acids, many of which are commercially available. The most preferred primary amine compound is tris(hydroxymethyl)aminomethane ("tris"), which is a commercially available buffering compound, and is particularly preferred because of its reactivity and its non-toxicity. This compound is sold under the trademark Trizma ® by Sigma Chemical Company of St. Louis, Missouri, for example. As discussed in more detail below, the volume of rinsing solution, the concentration of amine, and the number and duration of rinses used in the method of the present invention are interrelated, in that one advantageously should consider the other parameters in this group when selecting any one parameter in the group. For example, a decrease in any one (i.e., in the volume, the amine concentration, or the number or duration of the rinses) would generally reduce the efficiency of residual aldehyde removal from the tissue, but such a decrease may be offset by an increase in one or more of the other parameters. The amine compound that is attached to a solid support should be present in the rinsing solution in a concentration sufficient to react with substantially all of the residual free aldehyde groups in the tissue. Preferably, the concentration of the amine compound is such that there is a stoichiometric excess of the amino groups over the residual free aldehyde groups. Good results have been achieved using a rinsing solution containing more than a 100-fold excess of amino groups over the residual aldehyde groups. Preferably, the amine groups are present in at least a 5-fold excess over the residual free aldehyde groups. Residual aldehyde in aldehyde-treated bioprosthetic tissue may be measured by immersing a piece of the bioprosthetic tissue in a liquid such as saline for approximately 24 hours, then determining the amount of aldehyde in the solution by standard gas chromatography techniques. The amine concentration in the rinsing solution may vary considerably and still be effective in binding aldehydes leaching from biological tissue and promoting further diffusion of aldehydes out of the tissue according to the method of the invention. Advantageously, however, the amine concentration is high enough to promote removal of residual aldehyde from the tissue within a relatively short time. In general, the lower the concentration of a particular amine, the longer the total rinsing time will be. The amine concentration may also vary according to the ratio of tissue weight and surface area to solution volume. As the solution volume for rinsing a particular piece of tissue decreases, the amine concentration in the solution would have to increase to keep the total rinsing time the same. In addition, the amine concentration may vary according to the number of rinses (i.e., the number of fresh changes of amine solution in which the tissue is immersed), with the number of rinses required decreasing as the amine concentration increases. Likewise, if the rinsing solution is constantly replenished, a lower amine concentration may be used. As discussed above, the reactivity of the various amine compounds toward aldehydes varies. Therefore, higher concentrations of the less reactive amines would be used to reduce aldehyde levels to an acceptable level in a desirably short period of time. The effective amine concentration ranges from 0.001M to 1M, preferably about 0.1M. Most preferably, the rinsing solution comprises 0.1M tris buffer, pH 7.4. The rinsing solution should be buffered and the pH of the solution is to be maintained within a physiologically acceptable range, as described above. When tris is used as the amine, it also serves as a buffer. In accordance with the present invention, the biological tissue is immersed in a volume of rinsing solution, which, together with the amine concentration and number of rinses (as explained above), is sufficient to promote efficient diffusion of residual aldehyde out of the tissue. Thus, the volume of rinsing solution may vary. A lower aliphatic alcohol, such as ethanol, optionally may be added to the rinsing solution. However, residual alcohol in the tissue may cause cytotoxicity if it is not sufficiently removed from the tissue prior to use, so the rinsing solution preferably is alcohol-free. According to the invention the amine compound is immobilized by attachment to a non-toxic solid particulate support. The resulting solid material is packed into a column or bed through which a tissue rinsing solution is percolated. Preferably, a slurry of the solid immobilized amine material in a buffered solution is prepared, and the tissue is immersed in the slurry. The slurry then is circulated around the tissue by any suitable means, e.g., by automatic stirring devices. Several solid support materials having free amino-group-containing ligands bound thereto are available commercially. Examples of these materials are agarose-butylamine (e.g., AGBUTYLAMINE ® ), agarose-decylamine (e.g., AGDECYLAMINE ® ), agarose-ethylamine (e.g., AGETHYLAMINE®), agarose-adipic acid hydrazide, agarose to which 1,6-diaminohexane is coupled ([Sepharose 4B]-NH-(CH₂)₆ -NH₂, sold as AH-Sepharose 4B ® ), Arginine-Sepharose 4B ® (agarose having the amino acid arginine bound thereto), and Lysine-Sepharose 4B ® (lysine bound to agarose), all available from Pharmacia P-L Biochemicals in Piscataway, New Jersey. Sepharose 4B ® designates a highly porous beaded agarose, at a concentration of 4% in the commercial preparation. Other such materials include amino terminal agarose (e.g., Affi-Gel 102 ® , an amino terminal agarose gel with a hydrophilic spacer arm, having the formula: [agarose]-OCH₂ CONH(CH₂ )₂ NH₂ ) or aminoethyl polyacrylamide gels (e.g., aminoethyl Bio-Gel P-2 ® or P-150 ® , which have the formula: [polyacrylamide]-CONHCH₂ CH₂ NH₂ , where the polyacrylamide is Bio-Gel P-2 ® , 200-400 mesh, or Bio-Gel P-150 ® , 100-200 mesh.) These materials are available from Bio-Rad Laboratories, Richmond, California. Alternatively, the solid support material and an amine compound may be obtained separately, and the amine may be bound to the support by a variety of known techniques, depending on the particular amine and support chosen. The preparation of solid supports having desired compounds ("ligands") bound thereto is well known for use in affinity chromatography procedures. See, for example, Physical Biochemistry , 2nd Ed. , D. Freifelder, W.H. Freeman and Co., San Francisco, 1982, pp. 257-260. Many solid support materials having an amine compound attached thereto may be prepared for use in the method of the invention. The amine compound is bound to the support material, either directly or through a "spacer" compound, such that free amine groups remain after binding to the support is completed. The bond advantageously is non-reversible. The solid support material may be any suitable material which would not release toxic substances into the rinse solution. Such suitable materials include polyacrylamide and agarose, many types of which are commercially available from, for example, Pharmacia P-L Biochemicals, Piscataway, New Jersey, and BioRad Laboratories, Richmond, California. The linkage between the amine compound and the support may be accomplished by a variety of chemical reactions. One such known chemical reaction is cyanogen bromide activation of agarose, followed by reaction of the cyanogen bromide with amino groups on a ligand to bind the ligand to the solid support. Cyanogen bromide-activated agarose is commercially available (e.g., from Pharmacia P-L Biochemicals, Inc., Piscataway, New Jersey, U.S.A.) and readily couples primary amino groups. Therefore, diamine compounds (e.g., ethylenediamine, amino acids such as lysine or hydroxylysine, and many other compounds) can react with the activated agarose, and be bound thereto, and still have a free amino group. A polypeptide such as polylysine also may be coupled to the support, to provide a large number of free amine groups. The linkage of ligands to cyanogen bromide activated material is described in Wagner et al. ( Biochem. Biophys. Res. Commun. , 45 , 184 [1971]), and Lindberg and Persson ( Eur. J. Biochem. , 31 , 246 [1972]). Alternatively, a diamine compound may be attached to a solid support material having free carboxyl groups, through a carbodiimide reaction. Such solid supports include, among others, agarose having 6-amino-hexanoic acid coupled thereto ([Sepharose 4B]-NH-(CH₂ )₂-COOH, sold as CH-Sepharose-4B ® by Pharmacia P-L Biochemicals) and carboxymethyl agarose ([agarose]-OCH₂ COOH) or carboxyl terminal agarose ([agarose]-OCH₂ CONH(CH₂ )₂NHCO(CH₂)₂ COOH), both available from Bio-Rad Laboratories. The use of a carbodiimide coupling reagent (e.g., 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride) to link ligands to such supports is well known. To minimize possible steric hindrance of the reaction between the amino groups and the aldehyde compounds, the free amino groups attached to the solid support should not be too near the solid matrix surface. Thus, solid materials such as agarose-decylamine or AH-Sepharose 4B (described above), which have "spacers", comprising a carbon chain of ten and six carbon atoms, respectively, between the agarose surface and the free amino group, are generally preferred over such materials as agarose-ethylamine, which has only a 2-carbon spacer. One skilled in the art will recognize the wide variety of solid supports having ligands attached thereto which have free amino groups, which can be used in the method of the invention. According to this embodiment of the invention, the tissue is immersed in a rinsing solution which may be any suitable physiologically-compatible solution, such as distilled water, saline, phosphate-buffered saline, HEPES-buffered solution, or other buffered solution. To achieve the advantages of the immobilized amine embodiment (discussed below), the rinsing solution should be amine-free. The rinsing solution flows over both the tissue and the immobilized amine (the amine attached to the solid support). The aldehyde which has diffused out of the tissue into the rinsing solution therefore comes into contact with the immobilized amine, and is bound by the amine. The immobilized amine therefore removes free aldehyde from the solution, thereby promoting diffusion of additional aldehyde out of the tissue. The immobilized amine advantageously is in the same container as the tissue, with the immobilized amine material added to the rinsing solution to form a slurry. Alternatively, the immobilized amine, which may be packed into a column or bed, may be in a separate container connected, e.g., by tubing, to the container into which the tissue is placed, and the rinsing solution is cycled through both containers. If the tissue is sterilized prior to the rinsing procedure, the tissue, immobilized amine, and sterile rinsing solution all may be contained in a closed system, so that aseptic conditions can be maintained. The rinsing solution may be reused so long as reactive amino groups are present. A standard ninhydrin assay may be used to determine whether free amine has been depleted. Preferably, a large excess (e.g., 100-fold) of amine over aldehyde is used, so that the amine will not be depleted, and testing will not be necessary. The more often the solution is changed, and/or the larger the volume of solution contacting the tissue, the shorter the total rinsing time will be. Alternatively, the tissue may be immersed in a rinse solution which is constantly replenished, e.g., in a tank into which fresh solution is constantly added at the same rate at which solution is draining from the tank through an outlet. Rinsing continues until residual aldehyde levels have been decreased to a level at which the bioprosthetic tissue is substantially non-cytotoxic, as explained below. Preferably, residual aldehyde levels are decreased to about 1 part per million (ppm) or less. Residual aldehyde levels can be measured by any known means, including gas chromatography. For example, a sample of tissue rinsed in tris according to the method of the present invention may be transferred to saline solution for 24 hours, and the level of aldehyde which has leached into the solution is then measured by gas chromatography. The immobilized amine has the advantage over amine free in solution that neither the amine nor the amine-aldehyde complex actually comes into contact with the biological tissue, and therefore will not become entrapped within the tissue. Also, the amine will not bind to the tissue by reaction with reactive groups on the tissue. When aldehyde-treated tissue is treated according to the method of the present invention, residual aldehyde is decreased to levels significantly below the residual aldehyde levels in tissue rinsed with amine-free solutions, as demonstrated in the "Examples" below. The amount of rinsing time required to reduce residual aldehyde to an acceptable level is relatively short when the herein-disclosed method is employed, which is particularly beneficial to commercial enterprises in which bioprosthetic tissue is prepared on a large scale. The temperature of the rinsing solution may vary considerably. Slightly elevated temperatures may be employed advantageously, and may reduce the total rinsing time required, provided that the temperature is not so high as to cause deleterious effects on the tissue, however, the temperature is always maintained between 4°C and 45°C. Preferably, the temperature is about 35±2°C. The rinsing solution is preferably sterilized (by standard methods such as autoclaving) prior to contacting the tissue, and all rinsing steps are carried out under aseptic conditions. The bioprosthesis is then ready for implantation. Alternatively, the rinsed tissue may be stored until needed, preferably in 0.1M tris, pH 7.4, as described above. The following Examples are provided to illustrate specific embodiments of the method of the present invention. Extracted bovine ligaments are tanned by immersion in 0.625% (w/v) glutaraldehyde in an isotonic solution containing 5.39 g/l of the sodium salt of HEPES, 0.440 weight percent sodium chloride, and 2.6 g/l of MgCl₂·6H₂O at room temperature. Agarose-decylamine (AGDECYLAMINE ® , which is [Sepharose 4B]-NH-(CH₂)₂-NH₂) is purchased from Pharmacia P-L Biochemicals, Milwaukee, Wisconsin. A slurry of the agarose-decylamine is prepared by adding HEPES buffer to the commercially prepared suspension. The ligament tissue prepared above is immersed in the slurry. The concentration of bound ligand (i.e., the amine compound) per ml of gel is specified by the agarose-decylamine manufacturer. An amount of gel sufficient to provide at least a 100-fold excess of free amine groups over the free residual aldehyde groups (in the tissue) is present in the slurry. The total volume of the slurry is approximately 100 ml per 10 g (wet weight) of bioprosthetic tissue immersed therein. A magnetic stirring bar is added to the container to stir the slurry so it remains homogeneous and circulates over the tissue. The tissue is immersed in fresh slurry as needed. The tissue then is removed from the slurry, briefly rinsed (e.g., with saline or an aldehyde-free buffered solution) to remove residual agarose-decylamine material, and tested for cytotoxicity and residual glutaraldehyde as described below. Tissue samples having 1 ppm or less residual glutaraldehyde and which are non-cytotoxic may be sterilized by conventional techniques (excluding contact with aldehydes) or otherwise further processed in preparation for surgical implantation, by known techniques. If the tissue is not needed immediately for implantation, it is packaged and stored, as described below. A small amount of agarose-decylamine is added to the package, if desired. The samples were analyzed for cytotoxicity using an "agar overlay" method designed for cytotoxicity scoring of diffusible components in solid samples. The general procedure for the agar overlay test is described by Guess et al. ("Agar Diffusion Method for Toxicity Screening of Plastics on Cultured Cell Monolayers", J. Pharm. Sci. , 54:1545 [1965]). Two 1-centimeter pieces were cut from each ligament following the clinical saline rinses (or immediately after removal from the packaging solution for the six samples not clinically rinsed in saline) and were gently placed on an agar plate. Each agar plate had been previously prepared by cultivating mammalian cells thereon to form a cell monolayer on the agar surface, then staining the cells with a dye. After the ligament samples were placed on the agar plates, the plates were incubated at 37 ± 1°C in a humidified atmosphere containing 4-6% CO₂ for 24 ± 1 h. The plates then are examined microscopically at 100X for the percent of cells lysed in the vicinity of each ligament sample. Empty areas between cells are indicative of cell lysis. In addition to cytotoxicity evaluation, the ligaments were analyzed for residual glutaraldehyde, ethanol (for those samples contacted with ethanol during processing) and isopropanol (which contacted the samples during the sterilizing step). Pieces were cut from each ligament and immersed in saline solution (in separate containers). After 24 h at room temperature, the saline solution was analyzed for glutaraldehyde, isopropanol, and ethanol content using known gas chromotography procedures. Each of the 24 samples were found to have less than 1 part per million (ppm) residual glutaraldehyde. All but five of the samples had less than 1 ppm residual isopropanol, with the five samples having from 1.5 to 7.6 ppm isopropanol. Residual ethanol levels were found to be highest in samples contacted with ethanol during both "production rinse procedures" (i.e., the rinsing procedures according to the invention to remove residual aldehyde) and packaging, especially when rinsed by a "quick dip" in saline (as opposed to 3 X 5 min rinses in saline) after removal from the package.

Claims: A method for decreasing aldehyde levels in bioprosthetic tissue previously contacted with said aldehyde to one part per million (1 ppm) or less, comprising contacting said tissue with a rinsing solution, contacting and reacting the aldehyde which has diffused out of said tissue into said solution with an amine which is bound to a non-toxic solid support and which is contained in said rinsing solution in a concentration of from 0.001M to 1.0M, whereby the rinsing solution is maintained at a pH of between 7.0 and 7.6 and at a temperature of between 4°C and 45°C. The method of claim 1 wherein said biosprosthetic tissue is mammalian tissue selected from the group consisting of pericardial tissue, dura mater , fascia lata , amnion, tendon, ligament, cartilage, collagen, arteries, veins, skin patches and bone. The method of claim 2 wherein said biosprosthetic tissue is subjected to a calcification mitigation treatment. The method of claim 3 wherein said tissue is immersed in a rinsing solution, such that efficient diffusion of said aldehyde out of said tissue occurs, and said tissue is rendered substantially non-cytotoxic. The method of any of claims 1 to 3 wherein said rinsing solution is periodically or continuously contacted with said amine immobilized on said support. The method of claim 5 wherein said periodically changed rinsing solution is substantially free of unbound amine. The method of claim 5 wherein every 10 grams (wet weight) of said tissue is immersed in about 100mls of a slurry comprised of said amine immobilized on said support added to said rinsing solution.

Inventors: DIECK, RONALD, NASHEF, AWS
Applicants: BAXTER INTERNATIONAL INC. (A DELAWARE CORPORATION)
Application year: 1987
Main classification: A61L27
IPC codes: A61L27, A01N1