Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
  • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/505—Stabilizers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/005—Ingredients of undetermined constitution or reaction products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/143—Stabilizers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2400/00—Materials characterised by their function or physical properties
  • A61L2400/02—Treatment of implants to prevent calcification or mineralisation in vivo

Definitions

• Implantation of medical devices into humans and other animals is carried out with increasing frequency.
• Implantable medical devices may be formed wholly or partially of biological tissue that has been chemically "fixed” or preserved.
• the technique used for chemical fixation of biological tissues typically requires exposure of the biological tissue to one or more chemical agents that are capable of forming cross-linkages between connective tissue protein molecules present in the tissue.
• Examples of fixed biological tissues that have been used to form implantable bioprostheses include cardiac valves, blood vessels, skin, dura mater, pericardium, ligaments and tendons.
• the heart valve is one of the most widely studied implantable prosthesis and its clinical application and pathology are well documented (Schoen et al, Cardiovascular Pathology 1:29-52 (1992)). More than 100,000 cardiac valve prostheses are placed in patients each year (Levy, et al, U.S. Patent Number 5,674,29).
• the most common bioprosthetic heart valves in current use around the world are made of porcine aortic tissue or bovine pericardium tissue (Seifter, et al, U.S. Patent Number 5,476,516).
• Biological tissues including porcine aortic and bovine pericardium tissue, typically contain connective tissue proteins (i.e., collagen and elastin), which act as the supportive framework of the tissue.
• connective tissue proteins i.e., collagen and elastin
• the pliability or rigidity of each biological tissue is largely determined by the relative amounts of collagen and elastin present within the tissue and/or by the physical structure and conformation of its connective tissue frame work.
• Each collagen molecule is made up of three polypeptide chains intertwined in a coiled helical conformation.
• fixation To stabilize the bioprosthetic tissue, the reactive moieties of the tissue are blocked by process known as fixation. In 1968, it was found that collagen was stabilized by aldehydes (Nimni et al, J.
• Aldehydes stabilize collagen by forming chemical cross-linkages between the polypeptide chains within a given collagen molecule (i.e., intramolecular crosslinkages), or between adjacent collagen molecules (i.e., intermolecular crosslinkages).
• various chemical fixative agents sometimes referred to as tanning agents are known in the art.
• Fixative agents that have been utilized to cross-link collagenous biological tissues include aldehydes (e.g., formaldehyde, glutaraldehyde, dialdehyde starch, para formaldehyde, glyceraldehyde, glyoxal acetaldehyde, acrolein), dusocyanates (e.g., hexamethylene diisocyanate), carbodiimides, and certain polyepoxy compounds (e.g., Denacol-810, -512, or related compounds). Photooxidation has also been used to fix biological tissue.
• aldehydes e.g., formaldehyde, glutaraldehyde, dialdehyde starch, para formaldehyde, glyceraldehyde, glyoxal acetaldehyde, acrolein
• dusocyanates e.g., hexamethylene diisocyanate
• glutaraldehyde is used as the fixative for many commercially available bioprosthetic products, including porcine bioprosthetic heart valves (i.e., the Carpentier-EdwardsTM stented porcine bioprosthesis; Edwards Lifesciences, Irvine, Calif. 92614), bovine pericardial heart valve prostheses (e.g., Carpentier-EdwardsTM Perimount Pericardial Bioprosthesis, Edwards Lifesciences; Irvine, Calif. 92614) and stentless porcine aortic prostheses (e.g., EdwardsTM PRIMA Plus Stentless Aortic Bioprosthesis, Edwards Lifesciences, Irvine, Calif. 92614).
• porcine bioprosthetic heart valves i.e., the Carpentier-EdwardsTM stented porcine bioprosthesis; Edwards Lifesciences, Irvine, Calif. 92614
• bovine pericardial heart valve prostheses e.g., Carpentier-Edwards
• a primary failure mode of bioprosthetic tissue implantation including both porcine and pericardial biological replacement heart valves, is tissue calcification.
• tissue calcification causes more than 50 percent of heart valve bioprostheses failures within 10 years of implantation (Schoen et al, Cardiovascular Pathology 1:29-52 (1992)).
• calcification proceeds more rapidly in children than in adults (Id.).
• Calcification can result in undesirable stiffening or degradation of the bioprosthesis.
• Intrinsic calcification is characterized by the precipitation of calcium and phosphate ions within the fixed bioprosthetic tissue, including the collagen matrix and remnant cells.
• Extrinsic calcification is characterized by the precipitation of calcium and phosphate ions within the thrombus, including adherent cells (e.g., platelets) that adhere to the bioprosthesis and the development of calcium phosphate-containing surface plaques on the bioprosthesis.
• adherent cells e.g., platelets
• factors that affect the rate at which fixed tissue bioprostheses undergo calcification have not been fully elucidated.
• factors that are thought to influence the rate of calcification include: a) patient's age; b) existing metabolic disorders (i.e., c) hypercalcemia, diabetes, etc.); d) dietary factors; e) infection; f) parenteral calcium administration; g) dehydration; h) distortion/mechanical factors; i) inadequate coagulation therapy during initial period following surgical implantation; and j) host tissue responses.
• the factors that are thought to affect the propensity for platelets to adhere to a fixed bioprosthetic tissue include: a) tissue damage; b) diet; c) surface properties of the tissue, including the nature of exposed collagen (e.g., type I, IV, etc.); d) metabolic changes; e) coagulation; f) hemodynamics g) inflammation; and, h) infection.
• Various techniques have heretofore been proposed for reducing the in situ calcification of glutaraldehyde-fixed bioprostheses. Included among these calcification reduction techniques are the methods described in Nashef et al, U.S. Patent Number 4,885,005; Carpentier et al, U.S. Patent Number
• Figure 1 provides data from experiments to assess 90-day rat subcutaneous pericardial leaflet calcification ( ⁇ g Ca/mg dry wt) as described in Example 1 below.
• Figure 2 illustrates data from experiments to assess 90-day rat subcutaneous pericardial leaflet calcification ( ⁇ g Ca/mg dry wt) as described in Example 1 below.
• Figure 3 provides data from experiments to assess rat 90-day subcutaneous porcine leaflet calcification ( ⁇ g Ca/mg dry wt) as described in Example 2 below.
• Figure 4 illustrates data from experiments to assess 90-day rat subcutaneous porcine leaflet calcification ( ⁇ g Ca mg dry wt) as described in Example 2 below.
• the present invention provides methods of producing superior biological materials for implantation by mitigating calcification, reducing calcification, and/or reducing phospholipid content in the biological materials upon implantation.
• the procedures provide biological tissues that are well suited for use in bioprosthetic devices.
• the invention further provides superior biological materials prepared in accordance with the methods of the invention, and implantable prosthetics that comprise such superior biological materials.
• the present invention provides a method of treating a biological material, which comprises contacting the biological material with a preparation that contains a cross linking agent and a surfactant.
• the preparation does not contain a denaturant.
• the present invention provides a method of treating a biological material, which comprises the step of contacting the biological material with a preparation having a surfactant and a cross linking agent in the absence of a denaturant (hereinafter referred to as the SCL step); and the step of contacting the biological material with a preparation comprising a surfactant, a cross linking agent and a denaturant (hereinafter referred to as the SCLD step).
• the present invention provides a method of treating a biological material that comprises four separate steps performed in a specified sequence. In step one, the biological material is contacted with a preparation containing a cross linking agent in the absence of denaturant and surfactant.
• step two is performed, which comprises contacting the biological material with a preparation containing a cross linking agent and a surfactant in the absence of denaturant.
• step three is performed, which comprises contacting the biological material with a preparation containing a cross linking agent, a denaturant and a surfactant.
• the biological material is contacted with a terminal liquid sterilization solution.
• the present invention provides a method of treating a biological material that comprises contacting the biological material with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the method results in mitigation of calcification of the biological material upon implantation into a host organism relative to an identical method of treating the biological material with the exception that the biological material is not contacted with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the present invention provides a method of treating a biological material that comprises contacting the biological material with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the method results in reduction of phospholipid content of the biological material upon implantation into a host organism relative to an identical method of treating the biological material with the exception that the biological material is not contacted with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the present invention provides a method of treating a biological material that comprises contacting the biological material with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the method results in reduction of calcium content of the biological material upon implantation into a host organism relative to an identical method of treating the biological material with the exception that the biological material is not contacted with a preparation having a cross linking agent and a surfactant in the absence of a denaturant.
• the present invention provides a biological material resistant to calcification produced by a method of treating the biological material comprising the step of contacting the biological material with a preparation comprising a surfactant and a cross linking agent in the absence of a denaturant; and the step of contacting the biological material with a preparation comprising a surfactant, a cross linking agent and a denaturant.
• the present invention provides superior methods of treating a biological material, wherein the treatment mitigates calcification when the biological material is implanted into a host organism.
• the methods of treatment to mitigate calcification are also referred to herein as methods of mitigating calcification of a biological tissue.
• the methods of the present invention mitigate calcification of a biological material relative to methods that are identical with the exception that the SCL step is not performed. Individual steps of the methods of the present invention are described in detail below.
• Mitigation of calcification refers to reducing the incidence of calcification.
• the incidence of calcification refers to the number of members of a population of implanted biological materials for which calcification occurs relative to the total number of members of the population.
• Elimination of calcification means mitigating calcification wherein no members of a population of implanted biological materials are calcified.
• An exemplary calcification is the accumulation of calcium sufficient to cause stiffening or degradation of biological tissue implanted into a host organism.
• a variety of techniques are useful in measuring calcification in a biological material after implantation.
• calcification is measured by implanting the biological material in a host organism for a period of time, removing the implanted biological material, and measuring the calcium levels in the biological material.
• the biological material may be implanted subcutaneously or intramuscularly in, for example, a mammalian host organism between 10 and 100 days.
• the mammalian host organism may be any appropriate species, such as a rat or a rabbit.
• Another exemplary calcification is the measurement of calcium accumulation after implantation of the biological material into a mammalian host organism for 30 to 90 days.
• the biological material is then removed and analyzed for calcium content.
• biological material containing calcium levels below 5 ⁇ g of calcium per mg of dried material hereinafter referred to as ⁇ g Ca/mg
• ⁇ g Ca/mg biological material containing calcium levels below 5 ⁇ g of calcium per mg of dried material
• biological material containing calcium levels above 5 ⁇ g Ca/mg have inferior mechanical properties, decreased durability, and increased rates of failure when used as bioprosthetic tissue.
• bioprosthetic or biological tissue containing calcium content above 5 ⁇ g Ca/mg tissue is considered calcified whereas bioprosthetic or biological tissue containing calcium content below 5 ⁇ g Ca/mg tissue is considered not calcified.
• mitigation of calcification is defined as the reduction in the incidence of a biological material having greater than 5 ⁇ g Ca/mg dried material when implanted into a mammalian host organism for about 30 to about 90 days.
• calcification is measured by subcutaneously implanting the biological material into a rat for about 90 days.
• the bioprosthetic or biological tissue containing calcium above 5 ⁇ g Ca/mg tissue is considered calcified whereas bioprosthetic or biological tissue containing calcium below 5 ⁇ g Ca/mg tissue is considered not calcified.
• mitigation of calcification is defined as the reduction in the incidence of a biological material having greater than 5 ⁇ g Ca/mg material when implanted subcutaneously into a rat for about 90 days.
• mitigation of calcification results in elimination of calcification.
• the term "elimination of calcification” or “eliminating calcification” as used herein, means mitigating calcification wherein no members of a population of implanted biological materials are calcified.
• the present invention provides methods of treating a biological material, wherein the treatment reduces calcium content when the biological material is implanted into a host organism.
• the methods of treatment to reduce calcium content are also referred to herein as methods of reducing calcium content of a biological tissue.
• the methods of the present invention reduce calcium content of a biological material relative to methods that are identical with the exception that the SCL step is not performed. Individual steps of the methods of the present invention are described in detail below.
• METHODS OF REDUCING PHOSPHOLIPID CONTENT [0033]
• the present invention provides methods of treating a biological material, wherein the treatment reduces phospholipid content of the biological material.
• the methods of treatment to reduce phospholipid content are also refe ⁇ ed to herein as methods of reducing phospholipid content of a biological tissue.
• the methods of the present invention reduce phospholipid content of a biological material relative to methods that are identical with the exception that the SCL step is not performed. Individual steps of the methods of the present invention are described in detail below.
• Various methods of reducing phospholipid content in a biological material are useful in the current invention.
• phospholipid content of a biological material may be measured by extracting the tissue with a solvent mixture to solubilize the phospholipids.
• Exemplary phospholipids measured include phospholipids present in the tissue due to the cellular nature of the tissue.
• Phospholipid content of a biological material may be correlated to desired or undesired properties resulting from implantation of the biological material. For example, phospholipid content of a biological material may be measured and the biological material may then be implanted into a host organism. The properties of the biological material may then be analyzed. In an exemplary embodiment, phospholipid content is measured and the biological material is subsequently implanted into a mammalian host organism for 30 to 90 days.
• biological material containing phospholipid content below 1 ⁇ g of phospholipids per mg of dried material have superior mechanical properties, enhanced longevity and durability, and increased resistance to failure when used as bioprosthetic tissue compared to biological materials containing more than 1 ⁇ g PL/mg.
• biological material containing phospholipid content above 1 ⁇ g PL/mg have inferior mechanical properties, decreased durability, and increased rates of failure when used as bioprosthetic tissue.
• phospholipid content is measured and the biological material is subsequently implanted into a rat for about 90 days.
• desirable biological material contained less than 1 ⁇ g PL/mg and preferably less.
• biological material containing less than 0.4 ⁇ g PL/mg exhibits desirable properties after implantation into a rat for about 90 days.
• phospholipid content is measured by quantifying selected phospholipids within a sample.
• a variety of phospholipids may be selected for quantification, including acidic phospholipids, basic phospholipids and neutral phospholipids.
• the selected phospholipids are sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidic acid.
• the selected phospholipids include gangliotetraosylceramide (Gg4).
• the present invention provides methods of treating a biological tissue, wherein the treatment reduces phospholipid content of the biological tissue when implanted into a host organism.
• the methods of the present invention reduce phospholipids of a biological material relative to methods that are identical with the exception that the SCL step is not performed. Individual steps of the methods of the present invention are described in detail below.
• the methods of the present invention provide superior methods for mitigating calcification, reducing calcification, and/or reducing phospholipid levels in an implanted biological material.
• the methods of the present invention include the step of contacting the biological material with a preparation comprising a surfactant and a cross linking agent without denaturant (hereinafter referred to as the SCL step).
• the methods of the present invention include the step of contacting the biological material with a preparation comprising a surfactant, a cross linking agent and a denaturant (hereinafter refe ⁇ ed to as the SCLD step).
• the methods of the present invention further include, after completion of the SCL and SCLD steps previously discussed, contacting the biological tissue with a terminal liquid sterilization solution.
• the methods of the present invention further include, before performing the SCL and SCLD steps, contacting the biological tissue with a fixation solution that comprises a cross linking agent in the absence of denaturant.
• a fixation solution that comprises a cross linking agent in the absence of denaturant.
• Methods of the present invention include contacting a biological material with a surfactant, either alone or in combination with other chemical agents.
• the treatment can be performed as a single step. Alternatively, the treatment can be performed as a series of sequential steps. At the end of each sequential step, the surfactant is preferably removed and a new fraction of surfactant is added.
• Surfactants of use in the present invention include any surfactant, either substantially pure or containing additives.
• An exemplary surfactant is a surface active agent capable of reducing the surface tension of water. Exemplary defining features of surfactants can be found in KIRK- OTH ER'S "ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY," Third Edition, Vol. 22, pp. 332-432, which is hereby incorporated by reference.
• Nonionic surfactants include anionic, cationic, nonionic, and amphoteric surfactants.
• the method of the present invention uses a nonionic surfactant as the surfactant.
• Nonionic surfactants include, without limitation, various ethoxylates, carboxylic acid esters, glycol esters, polyoxyethylene esters, anhydrosorbitol esters, ethoxylated anhydrodrosorbitol esters, glycerol esters of fatty acids, carboxylic amides, diethanolamine condensates, and the like.
• Exemplary nonionic surfactants also include ethoxylated natural fats, oils and waxes.
• the skilled practitioner will recognize that many other surfactants are suitable for use in the method of the present invention. A list of additional surfactants can be found in U.S. Patent No. 6,214,054, which is incorporated by reference in its entirety for all purposes.
• the ethoxylated natural fats, oils, and waxes are optionally from a group including, without limitation, lauric, oleic, stearic, and palmitic fatty acids having trade names such as Armotan, Emsorb, Glycosperse, Hodag, and Tween.
• the surfactant is polyoxyethylene sorbitan monooleate (Tween 80).
• Surfactants such as Tween 80 are widely used in biochemical applications including: solubilizing proteins, isolating nuclei from cells in culture, growing of tubercule bacilli, and emulsifying and dispersing substances in medicinal and food products.
• the tissue is treated with a preparation or solution containing from about 0.1% to about 50% surfactant by weight. In another exemplary embodiment, the tissue is treated with a preparation or solution containing from about 0.1% to about 10% surfactant. In another exemplary embodiment, the tissue is treated with a preparation or solution containing more than 0.6% and less than 50% surfactant. In another exemplary embodiment, the preparation or solution contains from about 1.7% to about 25 % surfactant.
• the preparation or solution contains 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, or 2.9% surfactant.
• the preparation or solution contains 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% surfactant.
• Methods of the present invention include contacting a biological material with a cross linking agent, either alone or in combination with other chemical agents.
• the treatment can be performed as a single step.
• the treatment can be performed as a series of sequential steps.
• the cross linking agent is preferably removed and a new fraction of cross linking agent is added.
• exemplary cross linking agents include compounds capable of stabilizing the structure of a biological material through the formation of covalent bonds.
• Exemplary cross-linking reagents include, but are not limited to, aldehydes (e.g., formaldehyde, glutaraldehyde, dialdehyde starch, para formaldehyde, glyceraldehyde, glyoxal acetaldehyde, acrolein), dusocyanates (e.g., hexamethylene diisocyanate), carbodiimides (e.g.
• aldehydes e.g., formaldehyde, glutaraldehyde, dialdehyde starch, para formaldehyde, glyceraldehyde, glyoxal acetaldehyde, acrolein
• dusocyanates e.g., hexamethylene diisocyanate
• carbodiimides e.g.
• bifunctional pyridylthio compounds e.g. l,4-di-[3'-(2'-pyridylthio)- propionamido]butane
• bifunctional vinylsulfone compounds e.g. 1,6-hexane- bis-vinylsulfone
• photoactivatable cross-linkers e.g. azides, phenyl azides
• the cross linking agent is an aldehyde, including both mono- and poly-aldehydes.
• Aldehydes of use in practicing the present invention include any aldehyde, either substantially pure or containing additives.
• Exemplary aldehydes include one or more compounds of the following: acetaldehyde, butyraldehyde, isobutyraldehyde, propionaldehyde, ⁇ - methylpropionaldehyde, 2-methylbutyraldehyde, cyclopentanecarbaldehyde, benzaldehyde, caproaldehyde, carbaldehyde, glutaraldehyde, formaldehyde and the like.
• the tissue is treated with substantially any amount of aldehyde that provides the sought after results.
• the determination of the correct amount of aldehyde needed for a particular application is well within the abilities of those of skill in the art. For example, a biological tissue is contacted one or more times with aldehyde and the biological material is collected and analyzed.
• the cross linking agent is formaldehyde.
• formaldehyde A variety of formaldehyde concentrations are useful in the present invention.
• the tissue is treated with a formaldehyde solution containing from about 0.1% to about 50% formaldehyde by weight.
• the tissue is treated with a formaldehyde solution containing from about 1% to about 10% formaldehyde.
• the tissue is treated with a formaldehyde solution containing about 4% formaldehyde.
• the cross linking agent is glutaraldehyde.
• glutaraldehyde concentrations are useful in the present invention.
• the tissue is treated with a solution of glutaraldehyde containing from about 0.1% to about 50% of glutaraldehyde.
• the tissue is treated with a solution of glutaraldehyde containing from about 0.2% to about 3% of glutaraldehyde.
• An exemplary agent of use in the invention is a cross linking agent.
• a list of cross linking agents can be found in U.S. Patent No. 6,214,054, which is incorporated herein by reference in its entirety.
• Exemplary biological materials include those at least partially composed of a material of biological origin.
• tissue such as brain, muscle (including heart), liver, appendix, pancreas, gastiointestinal tract organs, skin, bone, cartilage, tendon, ligament, connective tissue, and lymphoid tissue such as thymus, spleen, tonsil, lymph nodes, and the like.
• the biological material may be a biological fluid.
• biological fluid includes, without limitation, to cerebrospinal fluid, blood, serum, plasma, milk, urine, saliva, tears, mucous secretions, sweat, semen and bodily fluids comprising these components.
• Bio materials may also include culture fluid (or culture medium) used in the production of recombinant proteins or containing cells in suspension prior to transplantation.
• biological materials include products made from human or animal organs or tissues, including serum proteins (such as albumin and immunoglobulin), hormones, food and processed food products, nutritional supplements, bone meal, animal feed, extracellular matrix proteins, gelatin, and other human or animal by products used in manufacturing or final goods.
• Biological materials may also refer to any material that can be found in a human or animal that is susceptible to infection or that may carry or transmit infection.
• the biological material is a bioprosthetic tissue.
• the bioprosthetic tissue may be incorporated into a bioprosthesis structure for implantation in the body.
• Bioprosthetic tissue may be comprised entirely of material of biological origin or may additionally comprise material of non-biological origin.
• One skilled in the art will readily recognize that various methods of assembling a bioprosthesis may be used to incorporate the bioprosthetic tissue into the bioprosthesis.
• the bioprosthetic tissue is a biological tissue.
• Biological tissues may be selected from a variety of tissue types, including substantially any mammalian tissue that is useful in preparing a bioprosthesis having a biological component thereto.
• the biological tissue is derived from an organ.
• the biological tissue is selected from nerve tissue, glandular tissue (e.g., lymphatic tissue), respiratory tissue, digestive tissue, urinary tract tissue, sensory tissue (e.g., cornea, lens, etc.), and reproductive tissue.
• glandular tissue e.g., lymphatic tissue
• respiratory tissue e.g., respiratory tissue
• digestive tissue e.g., urinary tract tissue
• sensory tissue e.g., cornea, lens, etc.
• reproductive tissue e.g., a biological fluid
• addition of liquid is not likely to be necessary, unless to dilute the ionic strength of the biological fluid to permit miscibility of the extraction solvent.
• the biological tissue is selected from muscle tissue, adipose tissue, epithelial tissue and endothelial tissue.
• the biological tissue is selected from myocardial tissue and vascular tissue.
• the biological tissue is selected from the group including, without limitation, heart valve, venous valve, blood vessel, aorta, artery, ureter, tendon, dura mater, skin, pericardium, intestine (e.g., intestinal wall),or periosteum.
• the biological tissue is derived from bone, cartilage (e.g. meniscus), tendon, ligament, or any other connective tissue.
• the biological tissue is porcine aortic tissue or bovine pericardium tissue.
• the source of the biological material used for this purpose may vary with regard to tissue type, the source may also vary with regard to species type (autologous, homologous or heterologous tissue).
• species type autologous, homologous or heterologous tissue.
• the biological material may first be suspended in an aqueous solution so that it will be suitable for use in the methods of the present invention.
• aqueous solution e.g. 0.32 M sucrose
• Other hypotonic or isotonic solutions include 5% dextrose, phosphate buffered saline, tri-buffered saline, HEPES-buffered saline, or any of the foregoing buffers.
• the biological material in the aqueous solution can also be homogenized, ground, or otherwise disrupted to maximize contact between the treatment agents and the biological material.
• cross linking agents are used to maintain the structural integrity of the biological tissue.
• Structural integrity can be defined as the ability of tissue to perform its necessary biological function. The artisan will appreciate that the degree of structural integrity required for the tissue to perform its necessary function may vary among different types of tissues. Further, particular applications for which the tissue is used may require different levels of structural integrity.
• the methods of the present invention include the step of contacting the biological material with a preparation comprising a surfactant and a cross linking agent without denaturant (hereinafter referred to as the SCL preparation).
• a preparation comprising a surfactant and a cross linking agent without denaturant (hereinafter referred to as the SCL preparation).
• Biological materials, cross linking agents, and surfactants useful in the current invention are discussed above and are likewise useful here in the SCL preparation.
• more than one SCL step is performed.
• the SCL step is performed from 1 to 5 times.
• the biological material is contacted with the SCL preparation only once.
• the tissue is treated with a SCL preparation containing from about 0.1% to about 50% surfactant by weight.
• the tissue is treated with a SCL preparation containing from about 0.1% to about 10% surfactant.
• the tissue is treated with a SCL preparation containing more than 0.6%) and less than 50%) surfactant.
• the SCL preparation contains from about 1.7% to about 25 % surfactant.
• the SCL preparation contains 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6, 2.7, 2.8, or 2.9% surfactant.
• the SCL preparation contains 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% surfactant.
• the surfactant used in the SCL preparation is a nonionic surfactant.
• the nonionic surfactant is Tween 80.
• the SCL preparation contains 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, or 2.9% of Tween 80.
• the SCL preparation contains 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% of Tween 80.
• surfactants may be used in the SCL step of the present method. Determining acceptable surfactants and surfactant levels to be used is well within the abilities of those of skill in the art given the guidance of the teachings herein.
• the biological tissue is treated with a SCL preparation containing a aldehyde as the cross linking agent.
• the aldehyde is glutaraldehyde.
• glutaraldehyde concentrations may be used in the SCL preparation.
• the preparation contains from about 0.1% to about 50% glutaraldehyde by weight.
• the tissue is treated with a SCL preparation containing from about 0.2% to about 3% of glutaraldehyde.
• the SCL step is performed at any appropriate temperature selected to provide optimal calcification reduction, calcification mitigation, and/or phospholipid content reduction.
• the SCL step is performed at approximately a single temperature. In another exemplary embodiment, the SCL step is performed at more than one temperature.
• the SCL step is performed at approximately room temperature. In another exemplary embodiment, heat is applied to the SCL preparation. In another exemplary embodiment, the SCL step is performed at a temperature between about 25°C and 55°C. In another exemplary embodiment, the SCL step is performed between about 35°C and 55°C. In another exemplary embodiment, the SCL step is performed between about 35°C and 45°C. In another exemplary embodiment, the SCL step is performed between about 35°C and 40°C. In another exemplary embodiment, the SCL step is performed at a temperature of about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C.
• the SCL step is performed for a time selected to provide optimal calcification mitigation, calcification reduction or phospholipid content reduction.
• the biological material is contacted with the SCL preparation for about 1 day to about 20 days.
• the SCL step lasts from 5 days to about 15 days.
• the SCL step is at least about 12 days. In another exemplary embodiment, the SCL step is at least about 12 days and the SCL preparation comprises at least about 1.7% surfactant.
• the SCL step is performed at a pH selected to provide optimal calcification mitigation or phospholipid content reduction.
• the pH of the SCL solution is between about 6 and about 8.
• the pH is maintained at about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.
• the SCL step can be performed as a single step.
• the SCL step can be performed as a series of sequential steps.
• the SCL preparation is preferably removed from the tissue prior to contacting the tissue with a new fraction of SCL preparation.
• the methods of the present invention include the step of contacting the biological material with a preparation comprising a surfactant, a cross linking agent and a denaturant (hereinafter referred to as the SCLD preparation).
• the SCLD step may be performed at different stages in the methods of the current invention. In an exemplary embodiment, the SCLD step is performed after the SCL step. In another exemplary embodiment, the SCLD step is performed before the SCL step.
• the SCLD step may also be referred to as a bioburden reduction step. In an exemplary embodiment, more than one SCLD step is performed. In another exemplary embodiment, two SCLD steps are performed. In an exemplary embodiment where two SCLD steps are performed, the first SCLD step may be refe ⁇ ed to as an initial bioburden reduction step (iBReP) and the second SCLD step may be refe ⁇ ed to as a final bioburden reduction step (fBReP).
• iBReP initial bioburden reduction step
• fBReP final bioburden reduction step
• a variety of denaturants are useful in the SCLD step of the present invention. In an exemplary embodiment, the denaturant produces a reversible or i ⁇ eversible loss of higher order structure other than the primary structure of proteins in a biological material.
• the denaturant is a protic solvent useful in removing infectious materials or chemical agents.
• Protic solvents of use in practicing the present invention include water, alcohols, carboxylic acids, and the like.
• the denaturant is an alcohol or other solvent incorporating an alcohol. The biological material is treated with substantially any amount of alcohol that provides the sought after results.
• the determination of the co ⁇ ect amount of alcohol needed for a particular application is well within the abilities of those of skill in the art.
• a biological tissue is subjected to one or more SCLD steps and the biological material is collected.
• the amount of infectious material or chemical agent removed by the SCLD step is determined.
• the alcohol ceases to remove infectious agent or chemical agent from the tissue an end point is reached, which is indicative of the amount of alcohol necessary to remove the particular agent from the tissue.
• the tissue is treated with an aqueous alcohol solution containing from about 1% to about 100% alcohol, more preferably from about 10% to about 80% alcohol.
• Exemplary alcohols include one or more compounds of the group consisting of methanol, ethyl alcohol, propyl alcohol, butyl alcohol, pentyl alcohol, hexyl alcohol, heptyl alcohol, octyl alcohol, nonyl alcohol, decyl alcohol, long chain alcohols (e.g. 1,2-octanediol) and the like.
• a method of the present invention uses ethanol.
• the alcohol is isopropyl alcohol.
• the SCLD preparation comprises an aldehyde as the cross linking agent.
• formaldehyde is the cross linking agent.
• a variety of formaldehyde concentrations are useful in the SCLD preparation.
• the SCLD preparation comprises about 0.5% to about 10% formaldehyde.
• the SCLD preparation comprises about 4% formaldehyde.
• the SCLD preparation may employ any appropriate surfactant.
• the surfactant used in the SCLD preparation is a nonionic surfactant.
• the SCLD preparation comprises Tween 80 as the surfactant.
• the SCLD preparation comprises about 0.5% to about 5% Tween 80.
• the SCLD preparation comprises about 2.1% of Tween 80.
• the SCLD step is performed at a temperature selected to provide optimal calcification mitigation, calcification reduction and/or reduction of phospholipid content of a biological material.
• the SCLD step is performed at approximately a single temperature.
• the SCLD step is performed at more than one temperature.
• the SCLD step is performed at approximately room temperature. In another exemplary embodiment, heat is applied to the SCLD preparation. In another exemplary embodiment, the SCLD step is performed at a temperature between about 30°C and 55°C. In another exemplary embodiment, the SCLD step is performed between about 30°C and about 40°C.
• two SCLD steps are preformed: an iBReP step and an fBReP step.
• the iBReP step is performed at about room temperature and the fBReP step is performed at a temperature above room temperature.
• the fBReP step is performed between about 30°C and about 37°C.
• each SCLD step is performed for a time selected to provide optimal calcification mitigation, calcification reduction and/or reduction of phospholipid content of a biological material.
• the iBReP step is performed from about 1 hour to about 4 hours and the fBReP step is performed from about 5 hours to about 15 hours.
• the SCLD step is performed at a pH selected to provide optimal calcification mitigation, calcification reduction and/or reduction of phospholipid content of a biological material.
• the pH of the SCLD solution is between about 6 and about 8.
• the pH is maintained at about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9.
• the SCLD step can be performed as a single step. Alternatively, the extraction can be performed as a series of sequential steps. At the end of each sequential step, the SCLD preparation is preferably removed prior to contacting the tissue with a new fraction of the SCLD preparation. [0096] In an exemplary embodiment, the SCLD step is performed before the SCL step. In another exemplary embodiment, the SCLD step is performed after the SCL step.
• the methods of the present invention further include contacting the biological tissue with a terminal liquid sterilization solution.
• the biological tissue is contacted with a terminal liquid sterilization solution after completion of the SCL and SCLD steps previously discussed.
• Exemplary terminal liquid sterilization solutions are capable of sterilizing the biological material before implantation.
• Sterilants useful in the cu ⁇ ent invention include cross linking agents, heat, radiation, electron beams, ultraviolet radiation, and combinations thereof.
• the TLS solution comprises heat and a cross linking reagent. Exemplary cross linking agents useful in the TLS solution are described above.
• the TLS solution is heated to a temperature from about 35°C to about 50°C. In another exemplary embodiment, TLS solution is heated to a temperature from about 35°C to about 40°C.
• the TLS solution is maintained at a pH between about 6 and 9. In another exemplary embodiment, the TLS solution is maintained at a pH between about 7.2-7.5. [0101] In an exemplary embodiment, the TLS solution is contacted with the biological material for sufficient time to sterilize the biological material.
• the time required for sterilization will depend on the stringency of the TLS solution. For example, a TLS solution containing an aldehyde that is heated to about 37°C may require 1 to 6 days to sterilize the biological material whereas heating the TLS solution to 50°C may decrease the required time to 25 hours.
• the TLS solution comprises about 0.2%) to about 2.0% by weight glutaraldehyde.
• the terminal sterilization time and temperature is 1-6 days at 37 degrees Celsius. In another particularly preferred embodiment, the terminal sterilization time and temperature is 1-2 days at 50 degrees Celsius.
• the terminal sterilization time and temperature may be adjusted while maintaining the effectiveness of the TLS solution.
• the methods of the present invention further include contacting the biological tissue with a fixation solution that comprises a cross linking agent in the absence of denaturant.
• the biological tissue is contacted with the fixation solution before the SCL and SCLD steps, after the SCL and SCLD steps, or between the SCL and SCLD steps.
• the biological tissue is contacted with the fixation solution before the SCL and SCLD steps.
• Fixation solutions useful in the present invention include a cross linking agent to effect crosslinking of the connective tissue proteins within the tissue. Cross linking agents useful in the cu ⁇ ent invention are discussed above and are likewise useful here in the fixation solution.
• the fixation solution contains a cross linking agent at a concentration of about 0.2% to about 2.0 % by weight.
• the cross linking reagent is glutaraldehyde.
• the fixation solution contains glutaraldehyde as the cross linking agent at a concentration of about 0.2% to about 2.0 % by weight.
• the fixation solution is contacted with the biological material from about 0.5 hours to about 14 days.
• the fixation solution is contacted with the biological material for about 1-4 days.
• the fixation solution is contacted with the biological material for about 2 days.
• the fixation solution further comprises a surfactant. Useful surfactants and surfactant concentrations are described above and are equally applicable to the fixation solution.
• Example 1 describes a method of mitigating calcification in pericardial tissue wherein the pericardial tissue is subjected to treatment with a SCL preparation comprising 10% Tween 80 and 0.625% glutaraldehyde.
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques. [0112] After preparation of the bovine pericardial tissue leaflets, the samples were chemically fixed by immersion into a solution of 0.625% glutaraldehyde in aqueous solvent for 30 minutes followed by a 7 day quarantine.
• leaflets were treated with 0.625% glutaraldehyde and 10% Tween 80 in aqueous solvent for 7 days.
• Leaflets were then either subjected to subsequent steps or implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis.
• the leaflets that were implanted into rats are hereinafter referred to in Example 1 as "Post Tween Process" leaflets.
• Remaining leaflets were subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 to 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 to 37 degrees Celsius for 10 hours ⁇ 30 minutes.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 35-40°C (measured at representative coolest and warmest product internal location) for 25 - 35C.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter refe ⁇ ed to in Example 1 as "Tween/TLS Process" leaflets.
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques. [0121] After preparation, the leaflets were chemically fixed by treatment with 0.625% glutaraldehyde in aqueous solvent for 14 days. Leaflets were then either subjected to subsequent steps or implanted subcutaneously into Sprague- Dawley Rats for 90 days, explanted, and submitted for calcium analysis. The leaflets that were implanted into rats are hereinafter refe ⁇ ed to in Example 1 as "14-day Control" leaflets.
• Remaining leaflets were subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• Formaldehyde 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes. [0125] After treatment, the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 35 - 40°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours including.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter refe ⁇ ed to in Example 1 as "Full Process Control" leaflets.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days. The leaflets were removed and prepared for calcium content analysis.
• Leaflets were first lyophilized and weighed then treated with 70% Nitric Acid at 110°C until completely digested. The digested samples were brought to a final concentration of 3,000 ppm of KCl [0130] The samples were analyzed for calcium content by atomic absorption spectroscopy (AAS).
• AAS atomic absorption spectroscopy
• Figure 1 shows the total ⁇ g Ca/mg dry leaflet tissue for repetitive treatments using the four methods described above: 1) 14-day Control; 2) Full Process Control; 3) Post Tween Process; and 4) Tween/TLS Process.
• the data in Figure 1 shows a reduction in the average calcium content of the Tween/TLS Process and the Post Tween Process relative to the Full Process Control and 14- day Control.
• Figure 2 shows a diamond plot representation of the average calcium content decrease data in Figure 1.
• the top and bottom of the diamonds in Figure 2 form the 95% confidence interval for the means.
• the small squares are individual data points.
• the small horizontal lines near the top and bottom of each diamond are overlap lines.
• Table 1 shows the mitigation of calcification using the Post Tween/TLS step relative to the Full Process Control Leaflet tissue samples containing more than 5 ⁇ g Ca/mg dry leaflet tissue are calcified.
• Table 1 shows that 7.8% of the tissue from the Full Process Control (group 2) calcified while 0% from the Tween/TLS Process group (group 4) calcified.
• Example 2 describes a method of mitigating calcification in porcine heart valve tissue wherein the porcine heart valve tissue is subjected to treatment with a SCL preparation comprising 10% Tween 80 and 0.625% glutaraldehyde. 2.1. Methods
• Porcine heart valve tissue leaflets were prepared for chemical fixation by standard techniques. [0137] After preparation of the porcine heart valve tissue leaflets, the samples were chemically fixed by immersion into a solution of 0.625% glutaraldehyde in aqueous solvent for 7 days.
• leaflets were treated with 0.625% glutaraldehyde and 10% Tween 80 in aqueous solvent for 7 days. Leaflets were then either subjected to subsequent steps or implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis. The leaflets that were implanted into rats are hereinafter refe ⁇ ed to in Example 2 as "Post Tween Process" leaflets.
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 35 - 40°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours including heat up and cool down within 35 - 40°C.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter refe ⁇ ed to in Example 2 as "Tween/TLS Process" leaflets.
• Porcine heart valve leaf tissue were prepared for chemical fixation by standard techniques. [0146] After preparation, the leaflets were chemically fixed by treatment with 0.625% glutaraldehyde in aqueous solvent for 14 days. Leaflets were then either subjected to subsequent steps or implanted subcutaneously into Sprague- Dawley Rats for 90 days, explanted, and submitted for calcium analysis. The leaflets that were implanted into rats are hereinafter refe ⁇ ed to in Example 2 as "14-day Control" leaflets.
• Remaining leaflets were subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 37.5 ⁇ 2.5°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter referred to in Example 2 as "Full Process Control" leaflets.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days. The leaflets were removed and prepared for calcium content analysis.
• Leaflets were first lyophilized and weighed then treated with 70% Nitric Acid at 110°C until completely digested. The digested samples were brought to a final concentration of 3,000 ppm of KCl. [0155] The samples were analyzed for calcium content by atomic absorption spectroscopy (AAS).
• AAS atomic absorption spectroscopy
• Figure 3 shows the total ⁇ g Ca/mg dry leaflet tissue for repetitive treatments using the four methods described above: 1) 14-day Control; 2) Full Process Control; 3) Post Tween Process; and 4) Tween/TLS Process.
• the data in Figure 3 shows a reduction in the average calcium content of the Tween TLS Process relative to the Full Process Control and 14-day Control.
• Figure 4 shows a diamond plot representation of the average calcium content decrease data in Figure 3.
• the top and bottom of the diamonds in Figure 2 form the 95% confidence interval for the means.
• the small squares are individual data points.
• the small horizontal lines near the top and bottom of each diamond are overlap lines.
• Table 2 shows the mitigation of calcification using the Post Tween TLS step relative to the Full Process Control Leaflet tissue samples containing more than 5 ⁇ g Ca/mg dry leaflet tissue are calcified.
• Table 2 shows that 20% (7/35) of the tissue from the Full Process Control (group 2) calcified while 8.1% (3/37) from the Tween/TLS Process group (group 4) calcified.
• Example 3 describes a method of mitigating calcification in pericardial tissue wherein the pericardial tissue is subjected to treatment with a SCL preparation comprising 1.2% Tween 80 and 0.625% glutaraldehyde.
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.
• leaflets were then subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were freated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22%> ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• Formaldehyde 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes. [0168] After treatment, the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH. The volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 37.5 ⁇ 2.5°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter refe ⁇ ed in Example 3 as "Group 2" leaflets.
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.
• the samples were chemically fixed by immersion into a solution of 0.625% glutaraldehyde in aqueous solvent for 12-24 hours followed by a 7 day quarantine. Subsequently, the leaflets were treated with 0.625% glutaraldehyde and 1.2% Tween 80 in aqueous solvent for 7 days.
• leaflets were then subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22%) ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625%) glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4 % Formaldehyde, 22 % ethanol, 1.2 % Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes.
• the leaflets were rinsed in 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were treated at 35 - 40°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter referred to in Example 3 as "Group 3" leaflets. 3.1.4. Test Group, 1 Day Glutaraldehyde. 13 Day
• Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques. [0180] After preparation of the bovine pericardial tissue leaflets, the samples were chemically fixed by immersion into a solution of 0.625% glutaraldehyde in aqueous solvent for 12-24 hours. Subsequently, the leaflets were treated with 0.625%) glutaraldehyde and 1.2% Tween 80 in aqueous solvent for 13 days. [0181] The leaflets were then subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant.
• iBReP initial bioburden reduction process
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4 % Formaldehyde, 22 % ethanol, 1.2 % Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes.
• leaflets were rinsed in 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS). In the TLS procedure, leaflets were treated with a solution of 0.625 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets. Leaflets were treated at 35 40°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours.
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days, explanted, and submitted for calcium analysis and are hereinafter refe ⁇ ed to in Example 3 as "Group 4" leaflets. 3.1.3. Calcium Analysis
• leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days. The leaflets were removed and prepared for calcium content analysis.
• Leaflets were first lyophilized and weighed then treated with 70% Nitric Acid at 110°C until completely digested. The digested samples were brought to a final concentration of 3,000 ppm of KCl.
• Table 3 shows the mitigation of calcification using the 7 day Tween 80 test group (group 3) and 13 day Tween 80 test group (group 4) relative to the Full Process Control (group 2).
• Leaflet tissue samples containing more than 5 ⁇ g Ca/mg dry leaflet tissue are calcified.
• Table 2 shows that 4.26% (4/94) of the tissue from the Full Process Control (group 2) calcified while 0.0% (0/91) from the 7 day Glutaraldehyde, 7 day Tween 80 test group (group 3) calcified. In addition, only 2.25% of the 1 day Glutaraldehyde, 13 day Tween 80 test group (group 4) showed calcification. Thus, both group 3 and 4 showed a reduction in calcification relative to the Full Process Control groups.
• Example 4 describes a method of reducing phospholipid content in porcine heart valve tissue wherein the porcine heart valve tissue is subjected to treatment with a SCL preparation comprising between 0.6%-6% Tween 80 and 0.625% glutaraldehyde for 7 to 15 days.
• Porcine heart valve tissue leaflets were prepared for chemical fixation by standard techniques.
• Leaflets were then subjected to an initial bioburden reduction process (iBReP).
• iBReP initial bioburden reduction process
• leaflets were treated with a solution of formaldehyde-based sterilant.
• the solution contained 4% Formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer.
• the volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets.
• Leaflets were treated at room temperature for 2 - 2.5 hours.
• the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH.
• the volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 3 consecutive cycles of 10 minutes each.
• leaflets were subjected to a final bioburden reduction process (fBReP).
• fBReP bioburden reduction process
• Formaldehyde 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Leaflets were treated at 30 - 37 degrees Celsius for 10 hours ⁇ 30 minutes. [0199] After treatment, the leaflets were rinsed in 0.625% glutaraldehyde in phosphate buffer in aqueous solvent at 7.4 pH. The volume of the rinsing solution was equal to or greater than one hundred milliliters (100 ml) of solution per 3 leaflets. Rinsing was performed at room temperature for 4 consecutive cycles of 10 minutes each.
• leaflets were subjected to a terminal liquid sterilization process (TLS).
• TLS terminal liquid sterilization process
• leaflets were treated with a solution of 0.516 - 0.656 % glutaraldehyde in phosphate buffer in aqueous solvent at 7.2-7.5 pH. The volume of the solution was 100 ml of solution per 3 leaflets.
• Leaflets were freated at 35 - 40°C (measured at representative coolest and warmest product internal location) for 25 - 35 hours. 4.1.2. Phospholipid Analysis
• leaflets After undergoing the treatment outlined above, leaflets were implanted subcutaneously into Sprague-Dawley Rats for 90 days. The leaflets were explanted and prepared for phospholipid content analysis. [0202] Leaflets were frozen liquid in nitrogen, pulverized, and dissolved in extraction solvent containing chloroform and methanol. After addition of physiological saline, the precipitated proteins and bulk tissue were removed by centrifugation. The extracted lipids were then collected from the solvent phase and subjected to thin layer chromatography analysis.
• the selected phospholipids analyzed by TLC were sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidic acid.
• Table 4 shows the reduction of phospholipid content in methods using the SCL step relative to the method where no SCL step is performed.
• +++ represents O.200 ⁇ g PL/mg of dried tissue
• PL/mg of dried tissue + represents > 0.400 ⁇ g PL/mg of dried tissue and ⁇ 1.000 ⁇ g
• PL/mg of dried tissue represents >1.00 ⁇ g PL/mg of dried tissue

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Medicinal Chemistry (AREA)
• Oral & Maxillofacial Surgery (AREA)
• Dermatology (AREA)
• Transplantation (AREA)
• Vascular Medicine (AREA)
• Surgery (AREA)
• Heart & Thoracic Surgery (AREA)
• General Chemical & Material Sciences (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Botany (AREA)
• Materials For Medical Uses (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=30116063

Family Applications (1)

Country Status (7)

Families Citing this family (9)

Family Cites Families (8)

• 2003
  • 2003-07-16 EP EP03764694A patent/EP1531878A1/de not_active Withdrawn
  • 2003-07-16 US US10/620,712 patent/US20040093674A1/en not_active Abandoned
  • 2003-07-16 WO PCT/US2003/022121 patent/WO2004006974A1/en not_active Application Discontinuation
  • 2003-07-16 BR BR0305562-0A patent/BR0305562A/pt not_active IP Right Cessation
  • 2003-07-16 JP JP2004521862A patent/JP2005532877A/ja active Pending
  • 2003-07-16 AU AU2003249272A patent/AU2003249272A1/en not_active Abandoned
  • 2003-07-16 CA CA002490060A patent/CA2490060A1/en not_active Abandoned

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events