WO2013090878A1

WO2013090878A1 - Coronary artery bypass grafts and vascular conduites

Info

Publication number: WO2013090878A1
Application number: PCT/US2012/070003
Authority: WO
Inventors: Kemal Schankereli
Original assignee: Vascudyne, Llc
Priority date: 2011-12-15
Filing date: 2012-12-17
Publication date: 2013-06-20

Abstract

This invention relates to coronary artery bypass grafts and vascular conduits, and in particular, to specially modified vascular tissue used to create an anastomosis.

Description

PATENT APPLICATION

TITLE

[para 2] CORONARY ARTERY BYPASS GRAFTS AND VASCULAR CONDUITS

CROSS REFERENCE TO RELATED APPLICATIONS

[para 3] Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[para 4] No federal government funds were used in researching or developing this invention.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

[para 5] Not applicable.

SEQUENCE LISTING INCLUDED AND INCORPORATED BY REFERENCE HEREIN

[para 6] Not applicable.

BACKGROUND

[para 7] Field of the Invention

[para 8] This invention relates to coronary artery bypass grafts and vascular conduits, and in particular, to specially modified vascular tissue used to create an anastomosis.

[para 9] Background of the Invention

[para 10] Anastomosis is a procedure where two separate tubular or hollow organs are surgically grafted together to form a continuous fluid channel between them. Vascular anastomosis involves creating an anastomosis between blood vessels to create or restore blood flow. When a patient suffers from coronary artery disease (CAD), an occlusion or stenosis in a coronary artery restricts blood flow to the heart muscle. In order to treat CAD, the area where the occlusion occurs is bypassed to reroute blood flow by grafting a vessel in the form of a harvested artery or vein, or a prosthesis. Anastomosis is performed between a graft vessel and one or more target vessels in order to bypass the blocked coronary artery, circumvent the occlusion and restore adequate blood flow to the heart muscle. This treatment is known as a coronary artery bypass graft procedure (CABG).

[para 11] However, numerous problems exist with the use of a harvested artery, vein, or vascular prosthesis in the creation of an anastomosis. Thrombosis, or clotting, is known to be a particularly serious adverse consequence of surgical anastomosis. Stenosis, or narrowing, is another serious adverse consequence that arises from damage of vascular tissue during the creation of a surgical anastomosis.

[para 12] Accordingly, there exists a need for vascular grafts, and in particular coronary artery bypass grafts, that do not result in thrombosis, stenosis, or other complications caused by vascular injury, that may be used in conjunction with, but do not require, the use of pharmaceutical or therapeutic agents or drugs to achieve such affects.

[para 13] It would also be advantageous to develop coated implantable medical devices such as prosthetic vascular grafts, as well as processes for manufacturing them.

BRIEF SUMMARY OF THE INVENTION

[para 14] Specifically, the invention relates to the preparation of animal vascular tissue, arteries or veins, in which the tissue is cleaned, chemically cross-linked using cross-linking agents, and surface- coated with an anhydride compound using a non-aqueous method of exposing tissue to the anhydride, resulting in an improved bioprosthetic or implantable surgical material that is substantially non- antigenic, non-thrombogenic, resistant to calcification, and durable enough to be used in surgical applications, such as the creation of an anastomosis.

[para 15] In a preferred embodiment, there is provided a process for the preparation of bioprosthetic animal vascular tissue, comprising the step of coating at least one surface of a cross-linked vascular tissue with an anhydride compound using a non-aqueous process of exposing the vascular tissue to the anhydride, wherein the cross-linked vascular tissue is an artery or vein obtained from an animal source, and wherein the vascular tissue is cross-linked by cross-linking agents to establish chemical cross-links within collagen of the vascular tissue. [para 16] In other preferred embodiments, there is provided additional alternative non- limiting features including:

wherein the non-aqueous process of exposing the vascular tissue to the anhydride is washing the vascular tissue in an anhydride solution;

wherein the anhydride solution is substantially non-aqueous;

wherein the non-aqueous anhydride solution is a mixture of an organic acid anhydride and an organic acid;

wherein the non-aqueous anhydride solution is a mixture of succinic anhydride and succinic acid;

wherein the non-aqueous anhydride solution is succinic anhydride in dry pyridine;

wherein the animal source is human, bovine, porcine, ovine, or equine;

wherein the bovine, porcine, ovine, or equine source is an animal 30 days old or less;

wherein the bovine, porcine, ovine, or equine source is an animal 5 days old or less;

wherein the vascular tissue is cross-linked using a 0.1% to 2.0% solution of a cross-linking agent selected from the group consisting of aldehydes, epoxides, isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acyl azides;

wherein the vascular tissue is cross-linked using about a 0.2% glutaraldehyde solution and the cross-linked vascular tissue is radially compliant; and/or

wherein the vascular tissue is cross-linked using about a 1.0% to 2.0% glutaraldehyde solution and the cross-linked vascular tissue is radially non-compliant.

[para 17] In other preferred embodiments, there is provided a further step of pre-treating the vascular tissue before the vascular tissue is cross-linked, wherein pre-treating is comprised of dehydrating and digesting with a surfactant.

[para 18] In another preferred embodiment, there is provided wherein dehydrating comprises subjecting the vascular tissue to a hyperosmotic salt solution.

[para 19] In other preferred embodiments, there is provided a further step of carbon sputter-coating the anyhydride-coated cross-linked vascular tissue with a carbon compound. [para 20] In other preferred embodiments, there is provided a further step of bonding a chelating agent to the surface of the anyhydride-coated cross-linked vascular tissue.

[para 21] In another preferred embodiment, there is provided wherein the chelating agent contains magnesium.

[para 22] In another preferred embodiment, there is provided a bioprosthetic vascular tissue material made according to the process(es) described and/or claimed herein.

[para 23] In another preferred embodiment, there is provided, wherein the bioprosthetic vascular tissue material is trimmed and configured to an appropriate shape for a surgical purpose selected from the group consisting of: coronary artery bypass grafting; creation of a therapeutic fistula e.g. for hemodialysis vascular access; carotid endarterectomy; endoscopic coronary surgery; and peripheral surgery.

BRIEF DESCRIPTION OF THE FIGURES

[para 24] FIGURE 1 is a flowchart showing Process 1 of the present invention and the steps of obtaining the collagenous tissue source, washing, dehydration, vapor cross-linking, liquid-bath cross- linking, non-aqueous anhydride surface coating, and processing into surgically usable tissue material, [para 25] FIGURE 2 is a flowchart showing Process 2 of the present invention and the steps of obtaining the collagenous tissue source, washing, dehydration, vapor cross-linking, liquid-bath cross- linking, non-aqueous anhydride surface coating, tissue chelation, and processing into surgically usable tissue material.

[para 26] FIGURE 3 is a flowchart showing Process 3 of the present invention and the steps of obtaining the collagenous tissue source, washing, dehydration, vapor cross-linking, liquid-bath cross- linking, non-aqueous anhydride surface coating, carbon sputter-coating, and processing into surgically usable tissue material.

[para 27] FIGURE 4 is a flowchart showing Process 4 of the present invention and the steps of obtaining the collagenous tissue source, washing, dehydration, vapor cross-linking, liquid-bath cross- linking, non-aqueous anhydride surface coating, both chelating and carbon sputter-coating, and processing into surgically usable tissue material. [para 28] FIGURE 5 is an illustration of one preferred anhydride-collagen chemical reaction according to the present invention.

[para 29] FIGURE 6 is an illustration of two examples of coronary artery bypass grafts that can be formed using the modified tissue material of the present invention.

[para 30] FIGURE 7 is an illustration of one example of an anastomosis / vascular conduit between adjacent vascular structures.

[para 31] FIGURE 8 is an illustration of one example of a peripheral, non-coronary use of the modified tissue material of the present invention, here showing a vascular graft to the kidney from the iliac artery.

DETAILED DESCRIPTION OF THE INVENTION

[para 32] Uses and Anastomosis

[para 33] The human body has numerous vessels carrying fluid to essential tissues and areas for recirculation or excretion. When vessels become damaged, severed or wholly occluded due to physiological problems, certain sections must be bypassed to allow for the free and continuous flow of fluids. Anastomosis is performed for the purpose of connecting different conduits together to optimize or redirect flow. In cardiac surgery, anastomosis is done to bypass the occluded vessel by harvesting a member of an unobstructed vessel and joining it to the occluded vessel below the point of stenosis, [para 34] The common procedure for performing the anastomosis during bypass surgery requires the use of very small sutures, loupes and microsurgical techniques. Surgeons must delicately sew the vessels together being careful not to suture too tightly so as to tear the delicate tissue, thereby injuring the vessel which may then result in poor patency of the anastomosis.

[para 35] An anastomosis may be compliant or noncompliant. A noncompliant anastomosis is one in which the anastomosis opening in the target vessel is not substantially free to expand or contract radially. A noncompliant anastomosis may be formed with a one-piece or multiple-piece anastomosis device that compresses or otherwise controls tissue in the vicinity of the anastomosis to hold the graft vessel in place relative to the target vessel. Noncompliant anastomoses have been successful in certain situations. A compliant anastomosis is one in which the target vessel is substantially free to expand or contract circumferentially and longitudinally in proximity to the anastomosis site. A traditional sutured anastomosis is compliant, and for this reason some surgeons would prefer to utilize an anastomosis system that provides a compliant anastomosis, particularly between a graft vessel and the aorta or other source of arterial blood.

[para 36] Vascular Tissue

[para 37] However, blood vessels are very complex structures that perform a wide array of functions beyond moving blood from point A to point B. They must not leak. They must avoid thrombosis. They must avoid stenosis. They control blood pressure. And, they perform the exchange of nutrients, ions, oxygen, and other biologically important molecules.

[para 38] Arteries and veins, although both act as blood vessels, do not share the same construction. An artery is composed of three main layers of tissue. These layers, in cross-section, from outside to inside, comprise the tunica externa (formerly known as the tunica advantitia) (outer), tunica media (middle), and tunica intima (inner). The tunica externa is made up of connective tissue, e.g. collagen and elastin. The tunica media is made up of smooth muscle cells and elastic tissue. The muscle fibers run both longitudinally and radially. The tunica intima is made up of laminin and endothelial cells, [para 39] "Thrombosis" is caused when the smooth muscle cells are exposed to flowing blood which triggers the release of Tissue Factor, also called Platelet Tissue Factor, and which initially is responsible for the biochemical cascade resulting in coagulation. Accordingly, vascular grafts must take this into account. Common stapling, suturing, puncturing and other manipulation by surgeons during the deployment of the anastomosis are sufficiently traumatic to the graft or adjacent tissues that they can cause thrombosis.

[para 40] "Stenosis" (narrowing) of re-stenosis of a blood vessel is the secondary consequence of damage to a blood vessel. After the initial damage and clotting has occurred, a secondary response results in the proliferation of cells in the tunica intima.

[para 41] "Intimal hyperplasia" is the thickening of the Tunica intima of a blood vessel as a

complication of a reconstruction procedure or endarterectomy. Intimal hyperplasia is the universal response of a vessel to injury and is an important reason of late bypass graft failure, particularly in vein and synthetic vascular grafts. As described in related publications, e.g. U.S. Pat. 7,932,265, multiple processes, including thrombosis, inflammation, growth factor and cytokine release, cell proliferation, cell migration and extracellular matrix synthesis each contribute to the restenotic process. Accordingly, it would be advantageous to avoiding stenosis in a newly deployed vascular graft. [para 42] Mechanism of Vascular Injury

[para 43] While the exact mechanism of stenosis is not completely understood, the general aspects of the restenosis process have been identified. When smooth muscle cells and endothelial cells within the vessel wall become injured, a thrombotic and inflammatory response is initiated. A proliferative and migratory response in medial smooth muscle cells is provoked by cell derived growth factors such as platelet derived growth factor, basic fibroblast growth factor, epidermal growth factor, thrombin, etc., released from platelets, invading macrophages and/or leukocytes, or directly from the smooth muscle cells. These cells undergo a change from the contractile phenotype to a synthetic phenotype

characterized by only a few contractile filament bundles, extensive rough endoplasmic reticulum, Golgi and free ribosomes. Proliferation/migration usually begins within one to two days' post-injury and peaks several days thereafter.

[para 44] Daughter cells migrate to the intimal layer of arterial smooth muscle and continue to proliferate and secrete significant amounts of extracellular matrix proteins. Proliferation, migration and extracellular matrix synthesis continue until the damaged endothelial layer is repaired, at which time proliferation slows within the intima, usually within seven to fourteen days post-injury. The newly formed tissue is called neointima. The further vascular narrowing that occurs over the next three to six months is due primarily to negative or constrictive remodeling.

[para 45] Simultaneously with local proliferation and migration, inflammatory cells adhere to the site of vascular injury. Within three to seven days post-injury, inflammatory cells have migrated to the deeper layers of the vessel wall. In animal models employing either balloon injury or stent

implantation, inflammatory cells may persist at the site of vascular injury for at least thirty days.

Inflammatory cells therefore are present and may contribute to both the acute and chronic phases of restenosis.

[para 46] The Modified Tissue of the Present Invention

[para 47] The present invention provides for the use of modified tissue, chemically and/or

mechanically modified, that does not elicit a thrombogenic response in the first place, and which may be used for creation of therapeutic surgical anastomosis and/or therapeutic fistulas,

[para 48] "Collagen" is the most abundant protein in all animal tissue, and is the primary component of connective tissue. Collagen consists of a protein with three polypeptide chains, each containing approximately 1000 amino acids and having at least one strand of repeating amino acide sequence Gly- X-Y, where X and Y can be any amino acid but usually are proline and hydroxyproline, respectively. Collagen assembles into different supramolecular structures and has exceptional functional diversity, [para 49] Vascular tissue sources contemplated as within the scope of the present invention include porcine, ovine, or bovine animals 30 days old or less. In one preferred embodiment, the tissue specimen is taken from an porcine, ovine, or bovine animal that is not more than about 10 days old, and in a preferred embodiment about 5 days old.

[para 50] "Vascular tissue" refers to an artery or vein.

[para 51] "Cross-links" are bonds that link one polymer chain to another. They can be covalent bonds or ionic bonds. "Polymer chains" can refer to synthetic polymers or natural polymers, including proteins such as collagen. Examples of some common crosslinkers are the dimethyl suberimidate, formaldehyde and glutaraldehyde. Each of these crosslinkers induces nucleophilic attack of the amino group of lysine and subsequent covalent bonding via the crosslinker.

[para 52] "Surfactants" are wetting agents that lower the surface tension of a liquid, allowing easier spreading, and lower the interfacial tension between two liquids. The term surfactant is a blend of "surface active agent". Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups (their "tails") and hydrophilic groups (their "heads"). Therefore, they are soluble in both organic solvents and water. Surfactants are also often classified into four primary groups: anionic, cationic, non-ionic, and zwitterionic (dual charge). A non- limiting preferred surfactant contemplated herein is sodium laurel sulfate, although various other surfactants known to a person of ordinary skill in the art are also contemplated as within the scope of the invention,

[para 53] The term "exposing" refers to soaking the tissue in a fluid comprising the treatment agent for a period of time sufficient to treat the tissue. The soaking may be performed by, but is not limited to, incubation, swirling, immersion, mixing, or vortexing. [para 54] Collagen content

[para 55] The use of young animals, for example, "bobby calf (BC) tissue for prostheses provides a number of benefits over alternative tissue sources. Young, or "BC" animals are primarily used for the production of veal, meaning that a large and steady supply of tissue from such animals is available. BC tissue is known to be extremely thin and has a very high natural collagen content, providing the tissue both high strength and a variety of biocompatibility benefits, including low antigenicity,

thrombogenicity and calcification potential; high endothelialization; high suture retention; and high bursting strengths. As the animal ages, the natural collagen content of its tissue decreases, and these biocompatibility benefits also decrease.

[para 56] Increasing the collagen content of a given specimen of animal tissue, and simultaneously decreasing the presence of non-collagenous proteins in such tissue results in a heightened

biocompatibility of treated tissue samples. The high natural collagen content of BC tissue makes it an excellent source of tissue for collagen-enhancing treatment.

[para 57] Alternatively, adult tissue may also be subjected to the steps of the present invention for the manufacturing of a source of surgical tissue.

[para 58] Bovine, ovine, equine, and porcine sources are used to provide the base material. In one preferred embodiment, the tissue source is bovine. In one preferred embodiment, the tissue source is ovine. In one preferred embodiment, the tissue source is equine. In one preferred embodiment, the tissue source is porcine.

[para 59] Washing

[para 60] Treatment of vascular tissue for use in surgical transplantation may optionally begin with an isotonic saline wash at room temperature. Washing a tissue sample with a surfactant/water solution for a period of up to 24 hours can result in a 99: 1 post-treatment ratio of collagen to non-collagenous proteins in the tissue. Such a high ratio greatly enhances the effectiveness of later collagen cross- linking to further improve biocompatibility of the sample.

[para 61] Thinness of tissue used for surgical implants and grafts provides many benefits in surgery as the thickness of such material directly affects the size of any product or device made with such a material, for example an anastomosis. Further, the smaller size impacts the ease with which the material may be introduced into the human body, through catheterization or otherwise, as well as the ease of manipulation of the material after placement. A thinner sample means a lower gauge catheter, and easier intravenous or percutaneous insertion, and thus the ability to treat a higher percentage of the patient population requiring such an intervention.

[para 62] Dehydration

[para 63] Suspension in a hyperosmotic solution for a period of 30 minutes will substantially thin the tissue through partial dehydration. The term "dehydration" broadly refers to any method that removes the water from the tissue without denaturing the tissue. This includes, but is not limited to processes such as, by way of example, and not limitation, lyophilization, vacuum drying, air drying, or solvent- based drying such as, by way of example, and not limitation, exposing the tissue to various alcohol- based solutions.

[para 64] Cross-Linking

[para 65] Once dehydrated (optionally), the vascular tissue is ready for collagen cross-linking.

Unfixed or non-crosslinked animal tissue undergoes enzymatic degradation when implanted in human/ animal body. Usually such degradation is followed after a moderate to severe inflammatory response; presumably due to an immunological reaction to the foreign biological materials in the host body. Cross-linking is a process well known in the art for improving the biocompatibility of collagen in a piece of tissue prior to surgical implantation. Processes for collagen cross-linking currently known in the art have been limited to the "tanning", or submersion of a tissue sample in a wet bath containing a cross-linking agent, such as an aldehyde, as a solute.

[para 66] Vapor Cross-Linking

[para 67] An ideal primary method for cross-linking collagen in tissue comprises placing such tissue onto a pin frame such that the edges are held firmly in place. The frame and tissue sample are then placed into a chamber equipped with each of an inlet and outlet port for submission to a "vapor cross- linking" process. The inlet port is attached to a stoppered flask comprising each of an inlet and outlet port and containing a bolus of polyoxymethylene, which flask is gently heated as air flow is simultaneously initiated from the flask into the chamber containing the tissue sample, thereby producing formaldehyde (cross-linking agent) vapors which flood the chamber for a period of 10 minutes, after which time such vapors are evacuated from the chamber and the pin frame and tissue sample are removed intact therefrom.

[para 68] The use of heated polyoxymethylene to create formaldehyde vapor is superior to the known method of heating liquid glutaraldehyde, as the latter decreases the efficiency of the vapor delivery mechanism by releasing water vapor. Water vapor will swell the tissue material, whereas the use of a formaldehyde vapor results in a relatively anhydrous cross-linking process. By not allowing excess water during this phase of the process, the tissue material maintains its thin profile. However, gas cross-linking with formaldehyde is limited by the structural size of formaldehyde and the available of a single aldehyde group.

[para 69] Liquid-Bath Cross-Linking

[para 70] Alternatively (or consecutively), the tissue material is subjected to a liquid cross-linking bath. In one preferred embodiment, this is a liquid glutaraldehyde bath. Glutaraldehyde provides a further cross-linking that results in additional cross-links that formaldehyde cannot achieve. The presence of two aldehyde groups for cross-linking and the ability to be cross link over a distance since glutaraldehyde has a three-carbon chain connecting the two carbonyl moieties further strengthens the tissue material. In one non-limiting preferred embodiment, the pin frame and tissue sample are then transferred into an aqueous bath containing 1% 0.01M phosphate buffered glutaraldehyde and 10% isopropyl alcohol at a temperature of approximately 40 degrees C, and gently stirred for a period of not less than 24 hours, although variations of glutaraldehyde cross-linking are well known in the art and are considered within the scope of this step of the present invention.

[para 71] Combination Vapor and Liquid-Bath

[para 72] The combination of vapor formaldehyde cross-linking with wet glutaraldehyde cross-linking results in improved stability of the cross-links when compared to a sample subjected to the latter process alone, the combination of formaldehyde vapor and glutaraldehyde liquid appears to provide an additional benefit to the material that results from the inventive process. The bioprosthetic vascular tissue implant material of the present invention does not exhibit the immunogenic problems known in the art that accompanies glutaraldehyde cross-linked materials. Prior research has shown that glutaraldehyde can trigger a strong inflammatory reaction within a mammalian body, even including anaphylactic reactions. However, the material produced by the present inventive process is non- antigenic.

[para 73] It is believed that the use of glutaraldehyde alone in chemical cross-linking of tissue may create cross-linking that is susceptible to opening and releasing glutaraldehyde after implantation of the sample. The result of such degradation of the cross-links would be an increased risk of inflammation in and around the implanted tissue. In contrast, when pre-treating the sample with vapor formaldehyde via the method described herein, the formaldehyde may act as a reducing agent, creating cyclic pyridine molecules. The process of creating stable, non-reactive aromatics on the exposed surface of the collagen is believed to progress by nucleophilic attack by formaldehyde on the carbonyl of the glutaraldehyde-linked amine of the lysine, histidine, and/or arginine, improving the stability of the molecular structure of the sample and reducing the antigenicity of the sample compared to a sample treated with glutaraldehyde alone. A reduced inflammatory response and lower degree of capsule formation provides a distinct advantage.

[para 74] Surface Coating with Anhydride

[para 75] After the completion of wet cross-linking, the tissue sample is subjected to the step of coating/reacting the cross-linked vascular tissue with an anhydride compound. Coating/reacting the surface with anhydride is used to chemically modify the surface of the vascular tissue to reduce or eliminate thrombosis caused by the implant. In one preferred embodiment, succinic anhydride is contemplated in order to create a chemical bond to the amino acid residues of the collagenous tissue. Amino acid residues such as lysine, arginine or histidine can be reacted with the anhydride to modify the surface of the tissue, and/or modify the electric charge that the tissue presents to the blood flow, [para 76] In one embodiment, the present invention provides for a method for forming a surface composition comprised of the vascular tissue and an anhydride including the step of using a nonaqueous process of exposing the vascular tissue to the anhydride, wherein the cross-linked vascular tissue is an artery or vein obtained from an animal source, and wherein the vascular tissue is cross- linked by cross-linking agents to establish chemical cross-links within collagen of the vascular tissue, [para 77] Usually, in order to convert the amine function of an amino acid into an amide, the pH of the solution must be raised to 10 or higher so that free amine nucleophiles are present in the reaction system. However, amines are efficiently acylated by both cyclic and acyclic anhydrides in aqueous medium with sodium dodecyl sulfate (SDS) - without use of acidic or basic reagents. Various amines and anhydrides were reacted with equal ease. S. Naik, G. Bhattacharjya, B. Talukdar, B. K. Patel, Eur. J. Or . Chem., 2004, 1254-1260.

[para 79] Although excess water may result in swelling of the vascular tissue and increase the size of the implant, the pre-treatment by dehydration and crosslinking will reduce this. Additionally, nonaqueous methods for acylating the amino groups are well-known in the art. U.S. Pat. No. 4,294,241 discloses that succinic anhydride reacts with collagen replacing amino groups by carboxyl groups. The carboxyl groups contained in the molecule are susceptible to esterification by the standard reaction with acidified alcohol, e.g., reaction with anhydrous methanol acidified with HCl. In the above reactions the net isoelectric point of collagen can be controlled, either negative or positive, or completely

neutralized. See also, Green et al, Biochemistry Jour., Vol. 54, p.181-187 (1952). Green et al. showed that low temperature non-aqueous acylation of collagen may be conducted by exposing the collagen to a combination of an acid anhydride, e.g. acetic anhydride or succinic anhydride, and it's acid, e.g. acetic acid or succinic acid, without resorting to non-aqueous acylation chemistry that requires very high temperatures or very toxic reactants or catalysts. Also, Additionally, coating the collagen with an anhydride may be accomplished by the following. Native collagen is reacted with succinic anhydride in a water ice-bath (Fig 3.3). Specifically, collagen is subjected over sufficient time to 0.05% acetic acid solution, and the pH is adjusted to 10 in a NaOH solution, the temperature of the solution was maintained at 0-2 °C in an ice/water bath. Then, succinic anhydride is added, and the pH was maintained at 9-10 for lh. Succinylated collagen is then obtained. After washing several times with distilled water, succinylated collagen may be stored in PBS buffer solution.

H₂COOH

[para 80]

[para 81] Sputter-Coating with Carbon Compound

[para 82] The present invention is also directed to a method of coating the vascular tissue with a very thin layer of carbon. Methods are known in the art for coating implants with turbostratic carbon, e.g. planar graphitic carbon, see e.g. U.S. Pat. 5,370,684, Vallana et al. This carbon coating is firmly adherent to the tissue, thereby augmenting the implants biocompatible properties. The method of the present invention comprises subjecting a source of carbon to a plasma beam generated by triode sputtering under vacuum conditions. Ionization of an inert gas and generation of the plasma beam therefrom is achieved utilizing the apparatus disclosed in 5,370,684 and related patents, incorporated herein by reference. Carbon atoms sputtered off the target are directed to the substrate to thereby deposit a thin biocompatible film on the substrate. The desired density of the carbon deposited on the substrate (2.1 g/cm³ preferably 2.2 g/cm³) is achieved by operating the triode sputtering apparatus under the following conditions: Filament current 80-90 amps ; sputtering voltage 2000-3200 volts ; sputtering current 0.1-0.3 amp ; pressure 6xl0^~4 to 6xl0^~3 mbar . This method of depositing the highly dense carbon may be used regardless of the configuration of the substrate, e.g. flat or curved or undulating. Here, it is used on modified vascular tissue as described herein.

[para 83] Tissue Chelation

[para 84] In one embodiment, the tissue may have its surface chemically modified to have a chelating agent affixed thereto. Chelating agents are known to have an anti-coagulating effect as well as provide the possibility of reducing calcification of the glutaraldehyde cross-linked bioimplants. A preferred chelating agent is a compound that contains magnesium, such as mesotetraphenylporphorin magnesium chloride.

[para 85] Other suitable chelating agents that may be used include modified and/or covalently attached aminopolycarboxylic acids, such as EDTA (ethylenediaminetetraacetic acid) and EGTA

(bisaminoethyl-glycolethertetraacetic acid), as well as polymeric ether chelating agents such as the polyoxyethylenes, polyoxyglycols, and poly-glymes; other structural components which form similar shapes such as cyclic antibiotics, amino acid peptides, and wholly synthetic or biological compounds, such as modified fullerenes, dendrimers, polysaccharides, polynucleic acids, or other compounds capable of complexing divalent cations due to their three dimensional shape and ionic character. The principal action of the agents described is complexation of metal compounds, such as calcium and magnesium, through one or more electron-donating groups. Metal cations have several available orbitals for bond formation with complexing agents; therefore, the chelating agent can be monodentate (from the Latin word dentatus, meaning "toothed."), such as the chlorides, cyanides, hydroxides, or ammonia complexes, and mixed complexes may be formed from these. In addition, the ligand may be multidentate, or containing multiple teeth, which can contribute two or more electron pairs to a complex. Ethylenediamine, NH2CH2CH2NH2, is an exemplary bidentate ligand. Other useful members of the aminopolycarboxylic acid family include DCTA (trans-diaminocyclohexanetetraacetic acid), NTA (nitrilotriacetic acid), and DTPA (diethylenetriameinepentaacetic acid).

[para 86] Sterile preparations

[para 87] The product is then optionally sterilized by transferring it to an aqueous bath consisting essentially of a 2% buffered glutaraldehyde solution containing 10% isopropyl alcohol, and is soaked therein at 42 degrees C for a period of no less than 24 hours. Upon completion of sterilization, the tissue sample is removed from the pin frame.

[para 88] Finally, the tissue sample is packaged for transport in a container together with a sterilizing 0.65%, 0.01 M phosphate buffered glutaraldehyde solution, in which solution the tissue sample may either float freely or be held stationary by attachment to a mylar film. [para 89] Upon removal from packaging, the tissue sample may be trimmed, sutured or otherwise manipulated to form the size and shape necessary for any vascular tissue implantation surgery for which such tissue would be appropriate.

[para 90] Pharmacologic Agents for Combination Therapy

[para 91] Numerous agents have been suggested as anti-pro liferative agents in restenosis and have shown some activity in experimental animal models. Some of the agents which have been shown to successfully reduce the extent of intimal hyperplasia in animal models, include: heparin and heparin fragments, colchicine, taxol, angiotensin converting enzyme (ACE) inhibitors, angiopeptin, cyclosporin A, goat-anti-rabbit PDGF antibody, terbinafme, trapidil, tranilast, interferon-gamma, rapamycin, steroids, ionizing radiation, fusion toxins, antisense oligionucleotides and gene vectors. Antiproliferative action on smooth muscle cells in vitro has been demonstrated for many of these agents, including heparin and heparin conjugates, taxol, tranilast, colchicine, ACE inhibitors, fusion toxins, antisense oligionucleotides, rapamycin and ionizing radiation. Thus, agents with diverse mechanisms of smooth muscle cell inhibition may have therapeutic utility in reducing intimal hyperplasia,

[para 92] However, attempts in human patients to prevent restenosis by systemic pharmacologic means have thus far been unsuccessful. Neither aspirin-dipyridamole, ticlopidine, anti-coagulant therapy (acute heparin, chronic warfarin, hirudin or hirulog), thromboxane receptor antagonism nor steroids have been effective in preventing restenosis, although platelet inhibitors have been effective in preventing acute reocclusion after angioplasty. The platelet GP Il.sub.b/III.sub.a receptor, antagonist, Reopro TM. is still under study but Reopro TM. has not shown definitive results for the reduction in restenosis following angioplasty and stenting. Other agents, which have also been unsuccessful in the prevention of restenosis, include the calcium channel antagonists, prostacyclin mimetics, angiotensin converting enzyme inhibitors, serotonin receptor antagonists, and anti-pro liferative agents. These agents must be given systemically, however, and attainment of a therapeutically effective dose may not be possible; anti-proliferative (or anti-restenosis) concentrations may exceed the known toxic concentrations of these agents so that levels sufficient to produce smooth muscle inhibition may not be reached.

[para 93] Surgical Uses of Tissue [para 94] Specific examples of surgical procedures where the modified tissue is contemplated for use include coronary artery bypass grafting, creation of a therapeutic fistula e.g. for hemodialysis vascular access, carotid endardarectomy, and endoscopic coronary or peripheral surgeries.

[para 95] Incorporation of Insubstantial Variations under the Doctrine of Equivalents

[para 96] The references recited herein are incorporated herein in their entirety, particularly as they relate to teaching the level of ordinary skill in this art and for any disclosure necessary for the commoner understanding of the subject matter of the claimed invention. It will be clear to a person of ordinary skill in the art that the above embodiments may be altered or that insubstantial changes may be made without departing from the scope of the invention. Accordingly, the scope of the invention is determined by the scope of the following claims and their equitable Equivalents.

Claims

We claim:

1. A process for the preparation of bioprosthetic animal vascular tissue, comprising the step of coating at least surface of a cross-linked vascular tissue with an anhydride compound using a non-aqueous process of exposing the vascular tissue to the anhydride, wherein the cross-linked vascular tissue is an artery or vein obtained from an animal source, and wherein the vascular tissue is cross-linked by cross- linking agents to establish chemical cross-links within collagen of the vascular tissue.

2. The process of claim 1, wherein the non-aqueous process of exposing the vascular tissue to the anhydride is washing the vascular tissue in an anhydride solution.

3. The process of claim 2, wherein the anhydride solution is substantially non-aqueous.

4. The process of claim 3, wherein the non-aqueous anhydride solution is a mixture of an organic acid anhydride and an organic acid.

5. The process of claim 4, wherein the non-aqueous anhydride solution is a mixture of succinic anhydride and succinic acid.

6. The process of claim 4, wherein the non-aqueous anhydride solution is succinic anhydride in dry pyridine.

7. The process of claim 1, wherein the animal source is human, bovine, porcine, ovine, or equine.

8. The process of claim 7, wherein the bovine, porcine, ovine, or equine source is an animal 30 days old or less.

9. The process of claim 7, wherein the bovine, porcine, ovine, or equine source is an adult animal.

10. The process of claim 1, wherein the vascular tissue is cross-linked using a 0.1% to 2.0% solution of a cross-linking agent selected from the group consisting of aldehydes, epoxides, isocyanates, carbodiimides, isothiocyanates, glycidalethers, and acyl azides.

11. The process of claim 10, wherein the vascular tissue is cross-linked using about a 0.2% glutaraldehyde solution and the cross-linked vascular tissue is radially compliant.

12. The process of claim 10, wherein the vascular tissue is cross-linked using about a 1.0% to 2.0% glutaraldehyde solution and the cross-linked vascular tissue is radially non-compliant.

13. The process of claim 1, further comprising the step of pre-treating the vascular tissue before the vascular tissue is cross-linked, wherein pre-treating is comprised of dehydrating and digesting with a surfactant.

14. The process of claim 13, wherein dehydrating comprises subjecting the vascular tissue to a hyperosmotic salt solution.

15. The process of claim 1, further comprising the step of carbon sputter-coating the anyhydride- coated cross-linked vascular tissue with a carbon compound.

16. The process of claim 1, further comprising the step of bonding a chelating agent to the surface of the anyhydride-coated cross-linked vascular tissue.

17. The process of claim 16, wherein the chelating agent contains magnesium.

18. A bioprosthetic vascular tissue material made according to the process of claim 1.

19. A bioprosthetic vascular tissue material made according to the process of claim 15.

20. A bioprosthetic vascular tissue material made according to the process of claim 16.

21. The bioprosthetic vascular tissue material according to any of claims 18-20, wherein the bioprosthetic vascular tissue material is trimmed and configured to an appropriate shape for a surgical purpose selected from the group consisting of: coronary artery bypass grafting; creation of a therapeutic fistula e.g. for hemodialysis vascular access; carotid endardarectomy; endoscopic coronary surgery; and peripheral surgery.