The present invention relates to the field of treatment of defective human heart valves, and in particular, to replacement and repair of defective or damaged human heart valves using a substantially immunologically compatible heart valve from a non-human animal.
Heart valves are composed of fibrochondrocytes and an extracellular matrix of collagen and elastic fibers, as well as a variety of proteoglycans. Various synthetic and tissue based materials (the latter either from the recipient organism or from a different organism within the same species) have been used for forming heart valve replacements. Each have their advantages and disadvantages.
In the case of synthetic heart valves, it may be possible to modify advantageously the properties of the heart valves by altering the monomers and/or the reaction conditions of the synthetic polymers. Synthetic heart valves may be associated with thromboembolism and mechanical failure, however. See U.S. Pat. No. 4,755,593.
Tissue based heart valves may demonstrate superior blood contacting properties relative to their synthetic counterparts. Tissue based heart valves also may be associated with inferior in vivo stability, however. See U.S. Pat. No. 4,755,593.
Pericardial xenograft tissue valves have been introduced as alternatives to the synthetic and the tissue based valves described above. See Ionescu, M. I. et al., Heart Valve Replacement With The Ionescu-Shiley Pericardial Xenograft, J. Thorac. Cardiovas. Surg. 73; 31-42 (1977). Such valves may continue to have calcification and durability problems, however. See Morse, D, ed. Guide To Prosthetic Heart Valves, Springer-Verlag, New York, 225-232 (1985).
Accordingly, there is a need for mechanically durable, flexible heart valves replacements which are capable of contacting the blood and are stable in vivo.
Much of the structure and many of the properties of original heart valves may be retained in transplants through use of heterograft or xenograft materials, that is, heart valve from a different species than the graft recipient. For example, tendons or ligaments from cows or other animals are covered with a synthetic mesh and transplanted into a heterologous host in U.S. Pat. No. 4,400,833. Flat tissues such as pig pericardia are also disclosed as being suitable for heterologous transplantation in U.S. Pat. No. 4,400,833. Bovine peritoneum fabricated into a biomaterial suitable for prosthetic heart valves, vascular grafts, burn and other wound dressings is disclosed in U.S. Pat. No. 4,755,593. Bovine, ovine, or porcine blood vessel xenografts are disclosed in WO 84/03036. However, none of these disclosures describe the use of a xenograft for heart valve replacement.
Once implanted in an individual, a xenograft provokes immunogenic reactions such as chronic and hyperacute rejection of the xenograft. The term xe2x80x9cchronic rejectionxe2x80x9d, as used herein, refers to an immunological reaction in an individual against a xenograft being implanted into the individual. Typically, chronic rejection is mediated by the interaction of IgG natural antibodies in the serum of the individual receiving the xenograft and carbohydrate moieties expressed on cells, and/or cellular matrices and/or extracellular components of the xenograft. For example, transplantation of heart valve xenografts from nonprimate mammals (e.g., porcine or bovine origin) into humans is primarily prevented by the interaction between the IgG natural anti-Gal antibody present in the serum of humans with the carbohydrate structure Galxcex11-3Galxcex21-4G1cNAc-R (xcex1-galactosyl or xcex1-gal epitope) expressed in the xenograft. K. R. Stone et al., Porcine and bovine cartilage transplants in cynomolgus monkey: I. A model for chronic xenograft rejection, 63 Transplantation 640-645 (1997); U. Galili et al., Porcine and bovine cartilage transplants in cynomolgus monkey: II. Changes in anti-Gal response during chronic rejection, 63 Transplantation 646-651 (1997). In chronic rejection, the immune system typically responds within one to two weeks of implantation of the xenograft.
In contrast with xe2x80x9cchronic rejectionxe2x80x9d, xe2x80x9chyperacute rejectionxe2x80x9d as used herein, refers to the immunological reaction in an individual against a xenograft being implanted into the individual, where the rejection is typically mediated by the interaction of IgM natural antibodies in the serum of the individual receiving the xenograft and carbohydrate moieties expressed on cells. This interaction activates the complement system, causing lysis of the vascular bed and stoppage of blood flow in the receiving individual within minutes to two to three hours.
The term xe2x80x9cextracellular componentsxe2x80x9d, as used herein, refers to any extracellular water, collagen and elastic fibers, proteoglycans, fibronectin, elastin, and other glycoproteins, which are present in heart valve.
Xenograft materials may be chemically treated to reduce immunogenicity prior to implantation into a recipient. For example, glutaraldehyde is used to cross-link or xe2x80x9ctanxe2x80x9d xenograft tissue in order to reduce its antigenicity, as described in detail in U.S. Pat. No. 4,755,593. Other agents such as aliphatic and aromatic diamine compounds may provide additional crosslinking through the side chain carboxyl groups of aspartic and glutamic acid residues of the collagen polypeptide. Glutaraldehyde and diamine tanning also increases the stability of the xenograft tissue.
Xenograft tissues may also be subjected to various physical treatments in preparation for implantation. For example, U.S. Pat. No. 4,755,593 discloses subjecting xenograft tissue to mechanical strain by stretching to produce a thinner and stiffer biomaterial for grafting. Tissue for allograft transplantation is commonly cryopreserved to optimize cell viability during storage, as disclosed, for example, in U.S. Pat. No. 5,071,741; U.S. Pat. No. 5,131,850; U.S. Pat. No. 5,160,313; and U.S. Pat. No. 5,171,660. U.S. Pat. No. 5,071,741 discloses that freezing tissues causes mechanical injuries to cells therein because of extracellular or intracellular ice crystal formation and osmotic dehydration.
The present invention provides a substantially non-immunogenic heart valve xenograft for implantation into a human in need of heart valve repair or replacement. The invention further provides methods for processing xenogeneic heart valve with reduced immunogenicity but with substantially native elasticity and load-bearing capabilities for xenografting into humans.
As used herein, the term xe2x80x9cxenograftxe2x80x9d is synonymous with the term xe2x80x9cheterograftxe2x80x9d and refers to a graft transferred from an animal of one species to one of another species. Stedman""s Medical Dictionary, Williams and Wilkins, Baltimore, Md. (1995).
As used herein, the term xe2x80x9cxenogeneicxe2x80x9d, as in, for example, xenogeneic heart valve, refers to heart valve transferred from an animal of one species to one of another species. Id.
The methods of the invention, include, alone or in combination, treatment with radiation, one or more cycles of freezing and thawing, treatment with a chemical cross-linking agent, treatment with alcohol or ozonation, and sterilization In addition to or in lieu of these methods, the methods of the invention include, alone or in combination, in any order, a cellular disruption treatment, glycosidase digestion of carbohydrate moieties of the xenograft, or treatment with proteoglycan-depleting factors. Optionally, the xenograft can be exposed to an aldehyde for further crosslinking. After one or more of the above-described processing steps, the methods of the invention provide a xenograft having substantially the same mechanical properties as a native heart valve.
As used herein, the term xe2x80x9ccellular disruptionxe2x80x9d as in, for example, cellular disruption treatment, refers to a treatment for killing cells.
In one embodiment, the invention provides an article of manufacture comprising a substantially non-immunogenic heart valve xenograft for implantation into a human.
In another embodiment, the invention provides a method of preparing a heart valve xenograft for implantation into a human, which includes removing at least a portion of a heart valve from a non-human animal to provide a xenograft; washing the xenograft in water and alcohol; and subjecting the xenograft to at least one treatment selected from the group consisting of exposure to ultraviolet radiation, immersion in alcohol, ozonation, and freeze/thaw cycling, whereby the xenograft has substantially the same mechanical properties as a corresponding portion of a native heart valve.
In yet still a further embodiment, the invention provides a xenograft formed of a soft tissue for implantation into a human comprising a portion of the soft tissue from a nonhuman animal, wherein the portion includes a plurality of extracellular components, a plurality of substantially only dead cells, and an aldehyde in an amount ranging from about 0.01% to about 5% crosslinking a plurality of proteins of the extracellular components, the extracellular components and the dead cells having substantially no surface carbohydrate moieties which are susceptible to glycosidase digestion, and whereby the portion is substantially non-immunogenic and has substantially the same mechanical properties as a corresponding portion of the native soft tissue, and wherein the soft tissue is suitable for use as heart valve xenograft material.
As used herein, the term xe2x80x9cportionxe2x80x9d refers to all or less than all of the respective soft tissue heart valve xenograft material. xe2x80x9cSoft tissue xenograft materialxe2x80x9d refers to the non-human heart valves, valve portions, such as leaftlets, and other soft tissue materials that can be fashioned into valves and valve portions, such as, for example, pericardium.
In another embodiment, the invention provides a method of preparing a heart valve xenograft for implantation into a human, which includes removing at least a portion of a heart valve from a non-human animal to provide a xenograft; washing the xenograft in water and alcohol; subjecting the xenograft to a cellular disruption treatment; and digesting the xenograft with a glycosidase to remove surface carbohydrate moieties, whereby the xenograft has substantially the same properties as a corresponding portion of a native heart valve. As used herein, the term xe2x80x9csurface carbohydrate moiety (moieties)xe2x80x9d refers to a terminal xcex1-galactosyl sugar at the non-reducing end of a carbohydrate chain.
In a further embodiment, the invention provides a method of preparing a heart valve xenograft for implantation into a human, which includes removing at least a portion of heart valve from a non-human animal to provide a xenograft; washing the xenograft in water and alcohol; subjecting the xenograft to a cellular disruption treatment; and digesting the xenograft with a proteoglycan-depleting factor to remove at least a portion of the proteoglycans from the xenograft, whereby the xenograft has substantially the same mechanical properties as a corresponding portion of a native heart valve and is substantially non-immunogemc.
In yet further embodiments, the invention provides articles of manufacture including substantially non-immunogenic heart valve xenografts for implantation into humans produced by one or more of the above-identified methods of the invention.
In another embodiment, the invention provides a heart valve xenograft for implantation into a human which includes a portion of a heart valve from a non-human animal, wherein the portion has substantially no surface carbohydrate moieties which are susceptible to glycosidase digestion, and whereby the portion has substantially the same mechanical properties as a corresponding portion of a native heart valve. In still yet another embodiment, the invention provides a heart valve xenograft for implantation into a human which includes a portion of a heart valve from a non-human animal, wherein the portion includes extracellular components and substantially only dead cells, the extracellular components having reduced proteoglycans. The portion of the heart valve is substantially non-immunogenic and has substantially the same mechanical properties as the native heart valve.
The present invention is directed against the chronic rejection of xenografts for implantation into humans. Accordingly, heart valve xenografts produced in accordance with the methods of the invention are substantially non-immunogenic, while generally maintaining the mechanical properties of a native heart valve.
While the heart valve may undergo some shrinkage during processing, a heart valve xenograft prepared in accordance with the invention will have the general appearance of a native heart valve xenograft. For example, a mitral valve xenograft prepared in accordance with the invention will have the general appearance of a native mitral valve, and semi-lunar valve xenografts of the invention will have the general appearance of a native semi-lunar valves. The heart valve xenograft may be valve segments, such as individual leaflets, each of which may be implanted into receipient heart.
The invention provides, in one embodiment, a method for preparing or processing a xenogeneic heart valve for engraftment into humans. The heart valve may be harvested from any non-human animal to prepare the xenografts of the invention. Heart valve from transgenic non-human animals or from genetically altered non-human animals may also be used as xenografts in accordance with the present invention. Preferably, bovine, ovine, or porcine hearts, and more preferably porcine hearts, serve as sources of heart valves used to prepare the xenografts. Alternatively, porcine pericardium can be used to form the heart valve xenografts of the present invention.
In the first step of the method of the invention, an intact heart is removed from a non-human animal. Suitable heart valve tissues are excised from the heart. Pericardium may be also harvested and implanted to replace or repair damaged heart valves by those of skill in the art using known techniques. Preferably heart valve from a corresponding heart valve is used to make the heart valve xenograft of the invention. For example, mitral valve may be used to make a mitral valve xenograft for implantation.
In accordance with the invention, the heart which serves as the source of the heart valve is collected from freshly killed animals and preferably immediately placed in a suitable sterile isotonic or other tissue preserving solution. Preferably, harvesting of the hearts and valves occurs as soon as possible after slaughter of the animal and preferably is performed in the cold, i.e., in the approximate range of about 5xc2x0 C. to about 20xc2x0 C., to minimize enzymatic degradation of the heart valve, under strict sterile technique.
The harvested valves and tissue are dissected free of adjoining tissue. Alternatively, a valve may be dissected with portions of the surrounding cardiac tissue. For example, tricuspid valves are excised as separate leaflets, or as an intact valve including the fibrous ring surrounding the auriculo-ventricular orifice and the tendinous chords. Once removed, optionally, the valve or valve portions are supported with stents, rings and the like. The heart valve or portion is carefully identified and dissected free of adhering tissue, plaques, calcifications and the like, thereby forming the xenograft.
In one form of the invention, porcine peritoneum or pericardium is harvested to form a heart valve xenografts according to procedures known to those of ordinary skill in the art. See, for example, the peritoneum harvesting procedure discussed in U.S. Pat. No. 4,755,593 by Lauren.
In a preferred form of the invention, the xenograft is then washed in about ten volumes of sterile cold water to remove residual blood proteins and water soluble materials. The xenograft is then immersed in alcohol at room temperature for about five minutes, to sterilize the tissue and to remove non-collagenous materials. After alcohol immersion, the xenograft may be directly implanted or may be subjected to at least one of the following treatments: radiation treatment, treatment with alcohol, ozonation, one or more cycles of freezing and thawing, and/or treatment with a chemical cross-linking agent. When more than one of these treatments is applied to the xenograft, the treatments may occur in any order.
In one embodiment of the method of the invention, the xenograft is treated by exposure to ultraviolet radiation for about fifteen minutes or gamma radiation in an amount of about 0.5 to 3 MegaRad.
In another embodiment, the xenograft is treated by again being placed in an alcohol solution. Any alcohol solution may be used to perform this treatment. Preferably, the xenograft is placed in a 70% solution of isopropanol at room temperature.
In still another embodiment, the xenograft is subjected to ozonation.
In a further embodiment of the method of the invention, the xenograft is treated by freeze/thaw cycling. For example, the xenograft may be frozen using any method of freezing, so long as the xenograft is completely frozen, i.e., no interior warm spots remain which contain unfrozen heart valve tissue. Preferably, the xenograft is dipped into liquid nitrogen for about five minutes to perform this step of the method. More preferably, the xenograft is frozen slowly by placing it in a freezer. In the next step of the freeze/thaw cycling treatment, the xenograft is thawed by immersion in an isotonic saline bath at room temperature (about 25xc2x0 C.) for about ten minutes. No external heat or radiation source is used, in order to minimize fiber degradation.
In yet a further embodiment, the xenograft optionally is exposed to a chemical agent to tan or crosslink the proteins within the extracellular components, to further diminish or reduce the immunogenic determinants present in the xenograft. Any tanning or crosslinking agent may be used for this treatment, and more than one crosslinking step may be performed or more than one crosslinking agent may be used in order to ensure complete crosslinking and thus optimally reduce the immunogenicity of the xenograft. For example, aldehydes such as glutaraldehyde, formaldehyde, adipic dialdehyde, and the like, may be used to crosslink the extracellular collagen of the xenograft in accordance with the method of the invention. Other suitable crosslinking agents include aliphatic and aromatic diamines, carbodiimides, diisocyanates, and the like.
When an aldehyde such as, for example, glutaraldehyde is used as the crosslinking agent, the xenograft may be placed in a buffered solution containing about 0.001% to about 5.0% glutaraldehyde and preferably, about 0.01 % to about 5.0% glutaraldehyde, and having a pH of about 7.4. More preferably about 0.01% to about 0.10% aldehyde, and most preferably about 0.01% to about 0.05% aldehyde is used. Any suitable buffer may be used, such as phosphate buffered saline or trishydroxymethylaminomethane, and the like, so long as it is possible to maintain control over the pH of the solution for the duration of the crosslinking reaction, which may be from one to fourteen days, and preferably from one to five days, and most preferably from three to five days.
Alternatively, the xenograft can be exposed to a crosslinking agent in a vapor form, including, but not limited to, a vaporized aldehyde crosslinking agent, such as, for example, vaporized formaldehyde. The vaporized crosslinking agent can have a concentration and a pH and the xenograft can be exposed to the vaporized crosslinking agent for a period of time suitable to permit the crosslinking reaction to occur. For example, the xenograft can be exposed to vaporized crosslinking agent having a concentration of about 0.001% to about 5.0% and preferably, about 0.01% to about 5.0%, and a pH of about 7.4. More preferably, the xenograft is exposed to the aldehyde in an amount ranging from about 0.01% to about 0.10%, and most preferably to an aldehyde ranging in an amount from about 0.01% to about 0.05%. The xenograft is exposed to the aldehyde for a period of time which can be from one to fourteen days, and preferably from one to five days, and most preferably from three to five days. Exposure to vaporized crosslinking agent can result in reduced residual chemicals in the xenograft from the crosslinking agent exposure.
The crosslinking reaction continues until the immunogenic determinants are substantially eliminated from the xenogeneic heart valve, but the reaction is terminated prior to significant alterations of the mechanical properties of the xenograft. When diamines are also used as crosslinking agents, the glutaraldehyde crosslinking occurs after the diamine crosslinking, so that any unreacted diamines are capped. After the crosslinking reactions have proceeded to completion as described above, the xenograft is rinsed to remove residual chemicals, and 0.01-.10 M glycine, and preferably, 0.01-0.05 M glycine is added to cap any unreacted aldehyde groups which remain.
In addition to the above treatments, the xenograft is subjected to a cellular disruption treatment to kill the xenograft""s cells. The cellular disruption treatment precedes or follows digestion of the xenograft with glycosidases to remove surface carbohydrate moieties from the xenograft. In addition or in lieu of the glycosidase treatment, either preceding or following the glycosidase treatment, the xenograft may be treated with proteoglycan-depleting factors.
The xenograft is subjected to a cellular disruption treatment to kill the cells of the heart valve. Typically after surface carbohydrate moieties have been removed from living cells and the extracellular components, the living cells reexpress the surface carbohydrate moieties. Reexpression of antigenic moieties of a xenograft can provoke continued immunogenic rejection of the xenograft. In contrast, dead cells are unable to reexpress surface carbohydrate moieties. Removal of antigenic surface carbohydrate moieties from dead cells and the extracellular components of a xenograft substantially permanently eliminates antigenic surface carbohydrate moieties as a source of immunogenic rejection of the xenograft.
Accordingly, in the above-identified embodiments, the xenograft of the present invention is subjected to freeze/thaw cycling as discussed above to disrupt, i.e., to kill the cells of the heart valve. Alternatively, the xenograft of the present invention is treated with gamma radiation having an amount of 0.2 MegaRad up to about 3 MegaRad. Such radiation kills the heart valve cells and sterilizes the xenograft. Once killed, the heart valve cells are no longer able to reexpress antigenic surface carbohydrate moieties such xcex1-gal epitopes which are factors in the immunogenic rejection of the transplanted xenografts.
Either before or after the heart valve cells are killed, in embodiments of the invention, the xenograft is subjected to in vitro digestion of the xenograft with glycosidases, and specifically galactosidases, such as xcex1-galactosidase, to enzymatically eliminate antigenic surface carbohydrate moieties. In particular, xcex1-gal epitopes are eliminated by enzymatic treatment with xcex1-galactosidases, as shown in the following reaction: 
The N/acetyllactosamine residues are epitopes that are normally expressed on human and mammalian cells and thus are not immunogenic. The in vitro digestion of the xenograft with glycosidases is accomplished by various methods. For example, the xenograft can be soaked or incubated in a buffer solution containing glycosidase. In addition, the xenograft can be pierced to increase permeability, as further described below. Alternatively, a buffer solution containing the glycosidase can be forced under pressure into the xenograft via a pulsatile lavage process.
Elimination of the xcex1-gal epitopes from the xenograft diminishes the immune response against the xenograft. The xcex1-gal epitope is expressed in nonprimate mammals and in New World monkeys (monkeys of South America) as 1xc3x97106-35xc3x97106 epitopes per cell, as well as on macromolecules such as proteoglycans of the extracellular components. U. Galili et al., Man, apes, and Old World monkeys differ from other mammals in the expression of xcex1-galactosyl epitopes on nucleated cells, 263 J. Biol. Chem. 17755 (1988). This epitope is absent in Old World primates (monkeys of Asia and Africa and apes) and humans, however. Id. Anti-Gal is produced in humans and primates as a result of an immune response to xcex1-gal epitope carbohydrate structures on gastrointestinal bacteria. U. Galili et al., Interaction between human natural anti-xcex1-galactosyl immunoglobulin G and bacteria of the human flora, 56 Infect. Immun. 1730 (1988); R. M. Hamadeh et al., Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces, 89 J. Clin. Invest. 1223 (1992). Since nonprimate mammals produce xcex1-gal epitopes, xenotransplantation of xenografts from these mammals into primates results in rejection because of primate anti-Gal binding to these epitopes on the xenograft. The binding results in the destruction of the xenograft by complement fixation and by antibody dependent cell cytotoxicity. U. Galili et al., Interaction of the natural anti-Gal antibody with xcex1-galactosyl epitopes: A major obstacle for xenotransplantation in humans, 14 Immunology Today 480 (1993); M. Sandrin et al., Anti-pig IgM antibodies in human serum react predominantly with Galxcex11-3Gal epitopes, 90 Proc. Natl. Acad. Sci. USA 11391 (1993); H. Good et al., Identification of carbohydrate structures which bind human anti-porcine antibodies: implications for discordant grafting in man. 24 Transplant. Proc. 559 (1992); B. H. Collins et al., Cardiac xenografts between primate species provide evidence for the importance of the xcex1-galactosyl determinant in hyperacute rejection, 154 J. Immunol. 5500 (1995). Furthermore, xenotransplantation results in major activation of the immune system to produce increased amounts of high affinity anti-Gal. In accordance with the invention, the substantial elimination of xcex1-gal epitopes from cells and from extracellular components of the xenograft, and the prevention of reexpression of cellular xcex1-gal epitopes diminish the immune response against the xenograft associated with anti-Gal antibody binding with xcex1-gal epitopes.
In addition, the heart valve xenografts of the invention may be treated with polyethylene glycol (PEG) prior to or concurrently with treatment with glycosidase. PEG acts as a carrier for the glycosidase by covalently bonding to the enzyme and to the collagen extracellular components. Further, PEG-treated xenografts reduce immunogenicity.
Either before or after the xenograft cells are killed, in embodiments of the invention, the xenograft is washed or digested with one or more different types of proteoglycan-depleting factors. The proteoglycan-depleting factor treatment can precede or follow glycosidase treatment. Proteoglycans such as glycosaninoglycans (GAGs) are interspersed either uniformly as individual molecules or within varying amounts within the extracellular components of the present invention""s xenograft. The GAGs include mucopolysaccharide molecules such as chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, dermatan sulfate, heparin sulfate, hyaluronic acid, and mixtures thereof. The proteoglycans including such GAGs contain attached carbohydrates such as xcex1-gal epitopes. Such epitopes stimulate an immune response once the xenograft is transplanted, as discussed above. Washing or digesting the xenograft with the proteoglycan-depleting factor removes at least a portion of the proteoglycans and attached xcex1-gal epitopes from the extracellular components of the xenograft, and thereby diminishes the immune response against the xenograft upon its transplantation. After the proteoglycan-depleting factor treatment and subsequent transplantation, natural tissue repopulates the remaining collagen shell.
Non-limiting examples of the proteoglycan-depleting factors used in the present invention include proteoglycan-depleting factors such as chondroitinase ABC, hyaluronidase, chondroitin AC II lyase, keratanase, trypsin, fibrinectin and fragments of fibronectin.
Other proteoglycan-depleting factors known to those of ordinary skill in the art are also possible for use with the present invention, however. The present invention""s xenograft is treated with proteoglycan-depleting factor in an amount effective for removing at least a portion of the proteoglycans from the extracellular components of the xenograft. Preferably, the xenograft is treated with proteoglycan-depleting factor such as hyaluronidase in an amount ranging from about 1.0 TRU/ml to about 100.0 TRU/ml or proteoglycan-depleting factor such as chondroitinase ABC in an amount ranging from about 0.01 u/ml to about 2.0 u/ml or most preferably, in an amount ranging from about 1.0 ul/ml to about 2.0 u/ml. The xenograft can also be treated with proteoglycan-depleting factor such as fibronectin fragment, (e.g., amino terminal 29-kDa fibronectin fragment) in an amount ranging from about .01 xcexcM to about 1.0 xcexcM, and preferably in an amount ranging from about 0.1 xcexcM to about 1.0 xcexcM.
Prior to treatment, the xenograft optionally may be pierced to increase permeability to agents used to render the xenograft substantially non-immunogenic. A sterile surgical needle such as an 18 gauge needle is used to perform this piercing step, or, alternatively a comb-like apparatus containing a plurality of needles are used. The piercing may be performed with various patterns, and with various pierce-to-pierce spacings, in order to establish a desired access to the interior of the xenograft. Piercing may also be performed with a laser. In one form of the invention, one or more straight lines of punctures about three millimeters apart are established circumferentially in the surface of the xenograft.
Prior to implantation, the heart valve xenograft of the invention may be treated with limited digestion by proteolytic enzymes such as ficin or trypsin to increase tissue flexibility, or coated with anticalcification agents, antithrombotic coatings, antibiotics, growth factors, or other drugs which may enhance the incorporation of the xenograft into the recipient. The heart valve xenograft of the invention may be further sterilized using known methods, for example, with additional glutaraldehyde or formaldehyde treatment, ethylene oxide sterilization, propylene oxide sterilization, or the like. The xenograft may be stored frozen until required for use.
The heart valve xenograft of the invention, or a segment thereof, may be implanted into damaged human hearts by those of skill in the art using known surgical techniques, for example, by open heart surgery, or minimally invasive techniques such as endoscopic surgery, and transluminal implantation. Specific instruments for performing such surgical techniques are known to those of skill in the art, which ensure accurate and reproducible placement of heart valve implants.