JP2005532877A - Improved biological material phospholipid reduction and calcification reduction - Google Patents

Abstract

There is a need in the tissue treatment field for effective calcification reduction, reduced calcification and phospholipid content of transplanted bioprosthetic tissue. The present invention provides an effective protocol for preparing biological tissue for incorporation as a bioprosthetic device. In a pre-transplant protocol, the biological tissue is treated with a surfactant and a cross-linking agent in the absence of a denaturing agent to reduce calcification of the biological tissue and phospholipid content The discovery that is reduced is disclosed herein. Moreover, biological tissue prepared under this protocol is well adapted for use in bioprosthetic devices.

Description

(Cross-reference of related applications)
This application claims the priority of US Provisional Patent Application No. 60 / 396,879 (filed July 16, 2002), assigned to the same person, which is hereby incorporated by reference in its entirety for all purposes. Incorporated herein by reference.

(Background of the Invention)
Implantation of medical devices into humans and other animals is becoming more frequent. Implantable medical devices can be formed in whole or in part from chemically “fixed” or stored biological tissue. Techniques used for the chemical fixation of biological tissue are typically to convert a biological tissue into one or more chemicals that can form crosslinks between connective tissue protein molecules present in the tissue. Requires exposure. Examples of fixed biological tissues that have been used to form implantable bioprostheses include heart valves, blood vessels, skin, dura mater, pericardium, ligaments and tendons.

  The heart valve is one of the most widely studied implantable prostheses, and its clinical application and pathology is well documented (Schoen et al., Cardiovascular Pathology 1: 29-52 (1992)). Over 100,000 heart valve prostheses are implanted in patients every year (Levy et al., US Pat. No. 5,674,29). The most common bioprosthetic heart valves currently used worldwide are made from porcine aortic tissue or bovine pericardial tissue (Seifter et al., US Pat. No. 5,476,516).

  Biological tissues, including porcine aortic tissue and bovine pericardial tissue, typically contain connective tissue proteins (ie, collagen and elastin) that serve as a supporting framework for the tissue. The flexibility and stiffness of each biological material is largely determined by the relative amount of collagen and elastin present in the tissue and / or by the physical structure and organization of the connective tissue framework. Each collagen molecule is composed of three polypeptide chains entangled in a coiled helical configuration.

  In order to stabilize the bioprosthetic tissue, the reactive part of the tissue is blocked by a process known as fixation. In 1968, it was found that collagen is stabilized by aldehydes (Nimni et al., J. Biol. Chem. 243: 1457-1466 (1968)). Aldehydes form collagen by forming chemical crosslinks between polypeptide chains in a given collagen molecule (ie, intramolecular crosslinks) or between adjacent collagen molecules (eg, intermolecular crosslinks). Stabilize. Today, various chemical fixatives (sometimes referred to as tanning agents) are known in the art. Fixatives that are used to crosslink collagenous biological tissues include aldehydes (eg, formaldehyde, glutaraldehyde, dialdehyde starch, paraformaldehyde, glyceraldehyde, glyoxalacetaldehyde, acrolein), diisocyanates (eg, Hexamethylene diisocyanate), carbodiimides, and certain polyepoxy compounds (eg, Denacol-810, Denacol-512, or related compounds). Photooxidation has also been used to fix biological tissues.

Among the various chemical fixatives that are available, glutaraldehyde is a commercially available bioprosthetic product (porcine bioprosthetic heart valve (ie, Carpentier-Edwards ^™ swine bioprosthesis; Edwards Lifesciences, Irvine, Calif. 92614). ), bovine pericardium heart valve prostheses ^{(e.g., Carpentier-Edwards TM Perimount pericardial Bioprosthesis} , Edwards Lifesciences; Irvine, Calif.92614), and no stents porcine arterial prosthesis ^{(e.g., Edwards TM PRIMA Plus Stentless Aortic Bioprosthesis} , Edwards L fesciences, Irvine, are used as a fixing agent for including Calif.92614)).

  The main cause of poor bioprosthetic tissue transplantation, including both porcine heart valves and pericardial biological replacement heart valves, is tissue calcification. For example, calcification makes more than 50% of heart valve bioprostheses worse within 10 years of transplantation (Schoen et al., Cardiovascular Pathology 1: 29-52 (1992)). Furthermore, calcification proceeds faster in children than in adults (Id). Morphological studies of explanted bioprostheses have shown that calcification often occurs in collagen fibrils, organelles (eg, mitochondria and inactivated cells (leaflet connective tissue cells, and muscle shelves and porcine aortic valve arterial walls). Nuclei (including cells), thrombus formation, and vegetation (Schoen FJ et al., J Card Surg 9 (Supplement): 222-227 (1994); Schoen FJ et al., J. Biomed. Mater). Res.47: 439-465 (1999); Ferrans YJ et al., Hum Pathol 18: 586-595 (1987)). Although the calcification process itself has been extensively studied, the mechanism of tissue calcification remains unclear.

  Calcification can result in undesirable stiffening or degradation of the bioprosthesis. Two types of calcification (endogenous and exogenous) are known to occur in fixed collagenous bioprostheses. Endogenous calcification is characterized by the precipitation of calcium and phosphate ions in fixed bioprosthetic tissues (including collagen matrix and residual cells). Exogenous calcification is characterized by the precipitation of calcium and phosphate ions in thrombus (including adherent cells (eg, platelets)) attached to the bioprosthesis and the appearance of calcium phosphate-containing surface plaques on the bioprosthesis .

Factors affecting the rate at which a fixed bioprosthesis undergoes calcification has not been fully elucidated. However, factors that are thought to affect the rate of calcification include the following:
a) patient age;
b) the presence of metabolic disorders (ie
c) hypercalcemia, diabetes, etc.);
d) dietary factors;
e) infection f) parenteral calcium administration;
g) dehydration;
h) distortion / mechanical factors;
i) Inappropriate coagulation treatment immediately after surgical implantation; and j) Host tissue response.

Factors that may affect the tendency of platelets to adhere to fixed biological tissue include the following:
a) tissue damage;
b) diet;
c) tissue surface properties (including exposed collagen properties (eg, type I, type IV, etc.));
d) metabolic changes;
e) coagulation;
f) hemodynamics;
g) inflammation; and h) infection.

  To date, various techniques have been reported to reduce in situ calcification of bioprosthesis immobilized with glutaraldehyde. These calcification reduction techniques include Nasef et al., US Pat. No. 4,885,005; Carpentier et al., US Pat. No. 4,648,881; Girardot et al., US Pat. No. 4,976,733; Carpentier et al. Pollock et al., EP 103947 A2; Nasef et al., US Pat. No. 4,885,005; Cunan et al., US Pat. No. 6,214,054, and Cunanan et al., US Pat. 09 / 693,186, which are incorporated herein by reference in their entirety for all purposes.

  However, there continues to be a need in the art for methods of producing bioprostheses that are resistant to undesirable stiffening or degradation. The present invention fulfills these and other needs.

(Summary of the Invention)
The present invention provides an excellent biological material for transplantation by reducing calcification, reducing calcification, and / or reducing phospholipid content in biological cells upon transplantation. Provide a method to generate. This procedure provides a biological tissue that is very suitable for use in a bioprosthetic device. The present invention further provides superior biological materials prepared according to the methods of the present invention, and implantable prostheses comprising such superior biological materials.

  Accordingly, in a first aspect, the present invention provides a method of treating a biological material, the method comprising contacting the biological material with a preparation comprising a cross-linking agent and a surfactant. To do. This preparation does not contain a modifier.

  In a second aspect, the present invention provides a method of treating a biological material, the method comprising treating the biological material with a preparation having a surfactant and a cross-linking agent and in the absence of a denaturing agent. Contacting the biological material with a preparation comprising a surfactant, a cross-linking agent and a denaturant (hereinafter referred to herein as the SCL step). , Called the SCLD process).

  In a third aspect, the present invention provides a method of processing biological material, which includes four separate steps performed in a specific order. In step 1, the biological material is contacted with a preparation comprising a cross-linking agent in the absence of a denaturant and a surfactant. After step 1, step 2 is performed, which includes contacting the biological material with a preparation comprising a crosslinking agent and a surfactant in the absence of a denaturing agent. After step 2, step 3 is performed, which includes contacting the biological material with a preparation comprising a crosslinker, a modifier and a surfactant. After step 3, the biological material is contacted with the final liquid sterilization solution.

  In a fourth aspect, the present invention provides a method of treating biological material, wherein the method comprises treating the biological material with a preparation having a crosslinker and a surfactant, and in the absence of a denaturing agent. The process of making it contact in is included. Compared to the same method of treating biological material, except that the biological material is not contacted with a preparation having a crosslinker and a surfactant in the absence of a denaturing agent, this method is Reduces calcification of biological material when implanted in an organism.

  In a fifth aspect, the present invention provides a method of treating a biological material, the method comprising in the absence of a biological material, a preparation having a crosslinker and a surfactant, and a denaturing agent. The process of making it contact in is included. Compared to the same method of treating biological material, except that the biological material is not contacted with a preparation having a crosslinker and a surfactant in the absence of a denaturing agent, this method is When transplanted into an organism, it reduces the phospholipid content of the biological material.

  In a sixth aspect, the present invention provides a method of treating biological material, wherein the method comprises treating the biological material with a preparation having a crosslinker and a surfactant, and in the absence of a denaturing agent. The process of making it contact in is included. Compared to the same method of treating biological material, except that the biological material is not contacted with a preparation having a crosslinker and a surfactant in the absence of a denaturing agent, this method is When transplanted into an organism, it reduces the calcium content of the biological material.

  In a seventh embodiment, the present invention provides a biological material resistant to calcification produced by a method of treating biological material, the method comprising: Contacting a preparation comprising an active agent and a cross-linking agent in the absence of a denaturing agent; and contacting the biological material with a preparation comprising a surfactant, a cross-linking agent and a denaturing agent. .

  These and other embodiments of the invention will be apparent from the detailed description below.

Detailed description of the method of the present invention to reduce or reduce calcification
In one aspect, the present invention provides an excellent method of treating biological material, where the treatment reduces calcification when the biological material is implanted into a host organism. Treatment methods for reducing calcification are also referred to herein as methods for reducing calcification of biological tissue. The method of the present invention reduces calcification of biological material compared to a method that is identical except that no SCL process is performed. The individual steps of the method of the invention are described in detail below.

  Reduction of calcification refers to a decrease in the occurrence of calcification. The occurrence of calcification refers to the number of members of the biological population when calcification occurs, as compared to the total number of members of the transplanted biological material population. The elimination of calcification means the reduction of calcification and the members of the transplanted biological material population are not calcified.

  An exemplary mineralization is the accumulation of sufficient calcium to cause stiffening or degradation of biological tissue transplanted into the host organism. Various techniques are useful in measuring calcification in biological material after transplantation. In a typical assay, calcification involves transplanting biological material into a host organism for a period of time, removing the implanted biological material, and measuring calcium levels in the biological material. Measured by. This biological material can be implanted, for example, subcutaneously or intramuscularly in a mammalian host organism for 10 to 100 days. The mammalian host organism can be any suitable species (eg, rat or rabbit).

  Another exemplary mineralization is the measurement of calcium accumulation after transplantation of biological material into mammalian host cells for 30-90 days. The biological material is then removed and analyzed for calcium content. Surprisingly, in this example, a biological material containing a calcium level below 5 μg / mg dry material (hereinafter referred to as μg Ca / mg) has more than 5 μg Ca / mg calcium. When used as a bioprosthetic tissue, it has superior mechanical properties, extended life and durability, and increased resistance to breakage compared to the biological material it contains. Conversely, biological materials containing calcium levels above 5 μg Ca / mg have inferior mechanical properties, reduced persistence, and increased failure rate when used as bioprosthetic tissue. Thus, in this example, bioprosthetic and biological tissues that contain calcium content above 5 μg Ca / mg tissue are considered calcified, while calcium content below 5 μg Ca / mg tissue Bioprostheses or biological materials containing are considered non-calcified. Thus, in this example, the reduction in calcification is as a decrease in the development of biological material having a calcium level greater than 5 μg Ca / mg dry material when implanted in a mammalian host organism for about 30-90 days. Defined.

  In another exemplary embodiment, calcification is measured by subcutaneous implantation of biological material into rats for about 90 days. A bioprosthesis or biological tissue containing calcium above 5 μg Ca / mg tissue is considered calcified, whereas a bioprosthesis or biological material containing calcium below 5 μg Ca / mg tissue Is considered not calcified. Thus, in this example, reduction in calcification is defined as a decrease in the development of biological material with calcium levels above 5 μg Ca / mg material when rats are implanted subcutaneously for about 90 days.

  In another exemplary embodiment, reducing calcification results in elimination of calcification. The terms “elimination of calcification” or “eliminating calcification” as used herein refer to a member of a population of transplanted biological material. It means reduction of calcification that is not calcified.

  In another aspect, the present invention provides a method for treating biological material, wherein the treatment reduces the calcium content when the biological material is implanted into a host organism. This method of treatment for reducing calcium content is also referred to herein as a method of reducing the calcium content of biological tissue. The method of the present invention reduces the calcium content of the biological material compared to the same method except that no SCL step is performed. The individual steps of the method of the invention are described in detail below.

(Method for reducing phospholipid content)
In another aspect, the present invention provides a method of processing a biological material, wherein the processing reduces the phospholipid content of the biological material. Treatment methods that reduce phospholipid content are also referred to herein as methods for reducing the phospholipid content of biological tissue. The method of the present invention reduces the phospholipid content of biological material as compared to a method that is identical except that no SCL step is performed. The individual steps of the method of the invention are described in detail below.

  Various methods for reducing the phospholipid content in biological materials are useful in the present invention. For example, the phospholipid content of a biological material can be measured by extracting tissue with a solvent mixture that dissolves the phospholipid. Examples of phospholipids to be measured include phospholipids present in tissues due to the cellular properties of the tissues.

  The phospholipid content of the biological material can be related to desired or undesired properties resulting from the implantation of the biological material. For example, the phospholipid content of a biological material can be measured and then the biological material can be implanted into a host organism. The properties of the biological material can then be analyzed. In an exemplary embodiment, phospholipid content is measured and the biological material is subsequently implanted into a mammalian host organism for 30-90 days. Surprisingly, in this example, a biological material with a phospholipid content containing 1 μg or less (hereinafter referred to as μgPL / mg) of phospholipid per mg of dry substance is 1 μg PL / mg. When used as a bioprosthetic tissue compared to biological materials containing more than mg, it has excellent mechanical properties, enhanced life and durability, and increased resistance to breakage. Conversely, biological materials containing the above 1 μg PL / mg phospholipid content have poor mechanical properties, reduced durability, and increased failure rate when used as bioprosthetic tissue.

  In another exemplary embodiment, the phospholipid content is measured and the biological material is then implanted into the rat over a period of about 90 days. In this example, the desired biological material contains less than 1 μg PL / mg and preferably little.

  In another exemplary embodiment, the biological material comprising less than 0.4 μg PL / mg exhibits the desired properties after implantation into a rat for about 90 days.

  In an exemplary assay, phospholipid content is measured by quantifying selected phospholipids in a sample. Various phospholipids can be selected for quantification, including acidic phospholipids, basic phospholipids, and neutral phospholipids. In another exemplary embodiment, the selected phospholipids are sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid. In another exemplary embodiment, the selected phospholipid comprises gangliotriosylceramide (Gg4).

  Accordingly, in another aspect, the present invention provides a method for treating biological tissue, wherein the treatment reduces the phospholipid content of the biological tissue when implanted in a host organism. The method of the present invention reduces phospholipids in biological materials as compared to methods that are identical except that no SCL step is performed. The individual steps of the method of the invention are described in detail below.

(Method of Invention)
The methods of the present invention provide an excellent method for alleviating calcification, reducing calcification, and / or reducing phospholipid levels in transplanted biological material. The method of the present invention includes a step (hereinafter referred to as an SCL step) of bringing a preparation containing a surfactant and a crosslinking agent into contact with a biological material without modification. In a further embodiment, the method of the invention includes a step of contacting a biological material with a preparation comprising a surfactant, a cross-linking agent and a denaturing agent (hereinafter referred to as the SCLD step).

  In an exemplary embodiment, the method of the present invention further includes contacting the biological material with the final liquid sterilization solution after completion of the SCL and SCLD steps discussed above.

  In another exemplary embodiment, the method of the present invention further comprises contacting the biological tissue with an immobilization solution containing a cross-linking agent without a denaturant prior to performing the SCL and SCLD steps. Including the step of

In another aspect, the present invention provides a method of processing biological material, which method comprises the following steps:
(A) first contacting the biological material with a preparation comprising a cross-linking agent;
(B) After step (a), a step of bringing a preparation containing a surfactant and a crosslinking agent into contact with a biological material (SCL step);
(C) after step (b), contacting the biological material with a preparation comprising a surfactant, a crosslinking agent and a denaturing agent (SCLD step); and (d) after step (c) Contacting the final liquid sterilization solution with the biological material.

  Illustrative examples of these and other steps are discussed in detail below.

(Surfactant)
The method of the present invention includes the step of contacting a surfactant (alone or in combination with other chemicals) with a biological material. Processing can be performed as a single step. Alternatively, this process can be performed as a series of continuous steps. At the end of each successive step, the surfactant is preferably removed and a fresh surfactant fraction can be added. Surfactants for use in the present invention include any surfactant, either substantially pure or containing additives. Exemplary surfactants are surfactants that can reduce the surface tension of water. Definitions of exemplary characteristics of surfactants can be found in KIRK-OTHMER'S “ENCYCLOPEDIIA OF CHEMICAL TECHNOLOGY”, 3rd edition, Vol. 22, pp 332-432 (which is incorporated herein by reference).

  Other exemplary surfactants include anionic, cationic, and amphoteric surfactants. In an exemplary embodiment, the method of the present invention uses a nonionic surfactant as the surfactant. Nonionic surfactants include, but are not limited to: various ethoxylates, carboxylic esters, glycol esters, polyoxyethylene esters, anhydrous sorbitol esters, ethoxylated anhydrous sorbitol esters, fatty acid glycerol Esters, carboxylic acid amides, diethanolamine concentrates, etc. Exemplary nonionic surfactants also include ethoxylated natural fats, oils, and waxes. Those skilled in the art will appreciate that many other surfactants are suitable for use in the methods of the present invention. A list of additional surfactants is found in US Pat. No. 6,214,054, which is incorporated by reference in its entirety for all purposes.

  Ethoxylated natural fats, oils, and waxes are optionally derived from the following groups, including but not limited to: lauric acid, oleic acid, stearic acid, and palmitic acid (Has trade names such as Armotan, Emsorb, Glycosperse, Hodag, and Tween).

  In another exemplary embodiment, the surfactant is polyoxyethylene sorbitan monooleate (Tween 80). Surfactants such as Tween 80 are widely used in the following biochemical applications: protein lysis, nucleic acid isolation from cultured cells, tuberculosis growth, and emulsification of substances in pharmaceuticals and foods. dispersion.

  In an exemplary embodiment, the tissue is treated with a preparation or solution containing about 0.1% to about 50% by weight surfactant. In another exemplary embodiment, the tissue is treated with a preparation or solution containing about 0.1% to about 10% surfactant. In another exemplary embodiment, the tissue is treated with a preparation or solution containing greater than about 0.6% and less than about 50% surfactant. In another exemplary embodiment, the tissue is treated with a preparation or solution containing about 1.7% to about 25% surfactant. In another exemplary embodiment, the preparation or solution is 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. In another exemplary embodiment, the preparation or solution is 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14 %, 15%, 16%, 17%, 18%, 19% or 20% surfactant.

(Crosslinking agent)
The method of the present invention includes contacting the cross-linking agent (alone or in combination with other chemicals) with the biological material. This process can be performed as a single step. Alternatively, this process can be performed as a series of continuous steps. At the end of each successive step, the crosslinker is preferably removed and a new crosslinker fraction is added. Exemplary cross-linking agents include compounds that can stabilize the structure of biological materials by the formation of covalent bonds.

  Exemplary crosslinkers include, but are not limited to: aldehydes (eg, formaldehyde, glutaraldehyde, dialdehyde starch, paraformaldehyde, glyceraldehyde, glyoxalacetaldehyde, acrolein), diisocyanates (eg, hexaxane). Methylene diisocyanate), carbodiimides (eg, dicyclohexyl carbodiimide, 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride), polyepoxy compounds (eg, Denacol-810, -512), bifunctional maleimide compounds (eg, 1,4-bis-maleimidyl-2,3-dihydroxybutane, N- (p-maleimidopropyloxy) succinimide ester), bifunctional N-hydroxy Succinimide ester compounds (eg, disuccinimidyl glutarate, 3,3′-dithiobis (sulfosuccinimidyl propionate)), bifunctional imide ester compounds (eg, dimethyl suberimidate, Functional pyridylthio compounds (eg 1,6-hexane-bis-vinylsulfone) and photoactivatable crosslinkers (eg azide, phenylazide).

  In an exemplary embodiment, the crosslinker is an aldehyde (including both monoaldehyde and polyaldehyde). Aldehydes used in the practice of the present invention include all aldehydes, either substantially pure or containing additives. Exemplary aldehydes include one or more of the following compounds: acetaldehyde, butyraldehyde, isobutyraldehyde, propionaldehyde, α-methylpropionaldehyde, 2-methylbutyraldehyde, cyclopentanecarbaldehyde, benzaldehyde, capro Aldehydes, carbaldehyde, glutaraldehyde, formaldehyde, etc.

  Those skilled in the art will appreciate that different cross-linking agents can be used in different processes of the present invention. The determination of acceptable crosslinkers used in each step of the present invention is well within the ability of one skilled in the art, given the guidance of the teachings herein.

  In an exemplary embodiment, the tissue is treated with substantially any amount of aldehyde that provides the required results. Determination of the exact amount of aldehyde required for a particular application is well within the ability of those skilled in the art. For example, the biological tissue is contacted with the aldehyde one or more times, and the biological material is recovered and analyzed.

  In an exemplary embodiment, the crosslinker is formaldehyde. Various formaldehyde concentrates are useful in the present invention. In an exemplary embodiment, the tissue is treated with a formaldehyde solution containing from about 0.1% to about 50% formaldehyde. In another embodiment, the tissue is treated with a formaldehyde solution containing about 1% to about 10% formaldehyde. In another embodiment, the tissue is treated with a formaldehyde solution containing about 4% formaldehyde.

  In another exemplary embodiment, the cross-linking agent is glutaraldehyde. Various concentrations of glutaraldehyde are useful in the present invention. In an exemplary embodiment, the tissue is treated with a glutaraldehyde solution containing about 0.1% to about 50% glutaraldehyde. In another exemplary embodiment, the tissue is treated with a glutaraldehyde solution containing about 0.2% to about 3% glutaraldehyde.

  Certain preferred embodiments of the present invention are illustrated by mitigating calcification from mammalian tissue through the use of formaldehyde or glutaraldehyde, but the focus on the use of these two aldehydes is explained And should not be construed as defining or limiting the scope of the invention.

  Those skilled in the art will appreciate that other agents are suitable for use in the methods of the invention. An exemplary agent for use in the present invention is a crosslinker. A list of crosslinking agents is found in US Pat. No. 6,214,054, which is incorporated by reference in its entirety.

(Biological material)
Exemplary biological materials include materials that are at least partially composed of materials of biological origin. Examples of biological materials of human or animal origin include tissues (eg brain, muscle (including heart), liver, appendages, pancreas, gastrointestinal organs, skin, bone, cartilage, tendon, ligament, connective tissue As well as lymphoid tissues (eg, thymus, spleen, tonsils, lymph nodes, etc.). Alternatively, the biological material can be a biological fluid. The term biological fluid includes, but is not limited to, cerebrospinal fluid, blood, serum, plasma, milk, urine, saliva, tears, mucus secretions, sweat, semen, and body fluids including these components. . Biological material can also include culture fluids (or culture media) used in the production of recombinant proteins, or culture fluids that contain cells in suspension prior to transplantation. In another exemplary embodiment, the biological material includes products made from human or animal organs or tissues (serum proteins (eg, albumin and immunoglobulins), hormones, food and processed foods, nutritional supplements Agents, bone meal, animal feed, extracellular matrix proteins, gelatin, and other human or animal by-products used in the manufacture or final product). Biological material can also refer to any material that can be found in a human or animal that is susceptible to infection or that can carry or transmit an infection.

  In another exemplary embodiment, the biological material is a bioprosthetic tissue. Bioprosthetic tissue can be incorporated into a bioprosthetic structure for implantation into the body. The bioprosthetic tissue may consist entirely of materials of biological origin and may further include materials of non-biological origin. Those skilled in the art will readily recognize that various methods of assembly of the bioprosthesis can be used to incorporate the bioprosthetic tissue into the bioprosthesis. In a further preferred embodiment, the bioprosthetic tissue is a biological tissue.

  The biological tissue can be selected from a variety of tissue types, including virtually any mammalian tissue useful in preparing a bioprosthesis having a biological component. For example, in one embodiment, the biological tissue is from an organ. In another embodiment, the biological tissue is selected from neural tissue, glandular tissue (eg, lymphoid tissue), respiratory tissue, digestive tissue, urinary tract tissue, sensory tissue (eg, cornea, lens, etc.), and reproductive tissue. Is done. However, in related embodiments where the biological tissue is a biological fluid, the addition of fluid is probably necessary unless the ionic strength of the biological fluid is diluted to allow the extraction solvent to be incorporated. is not.

  In an exemplary embodiment, the biological tissue is selected from muscle tissue, adipose tissue, epithelial tissue and endothelial tissue. In another exemplary embodiment, the biological tissue is selected from myocardial tissue and vascular tissue. In related embodiments, the biological tissue includes a heart valve, venous valve, blood vessel, aorta, artery, ureter, tendon, dura mater, skin, pericardium, intestinal tract (eg, intestinal wall), or periosteum. Is selected from the group not limited thereto. In another exemplary embodiment, the biological tissue is from bone, cartilage (eg, meniscus), tendon, ligament, or any other connective tissue. In another exemplary embodiment, the biological tissue is porcine aortic tissue or bovine pericardial tissue.

  Just as the source of biological material used for this purpose can vary with respect to the tissue type, this source can also vary with respect to the type of species (self-tissue, allogeneic tissue or heterogeneous tissue). The artisan will appreciate that the methods of the invention can be used with bioprosthetic devices that include one or more types of tissues or materials.

  In an exemplary embodiment where the biological material is a solid tissue or product, the material can first be suspended in an aqueous solution so as to be suitable for use in the methods of the invention. For example, brain tissue can be suspended in a sucrose solution (eg, 0.32 M sucrose) at 10% by weight to volume. Other hypotonic or isotonic solutions include 5% glucose, phosphate buffered saline, tris buffered saline, HEPES buffered saline, or any of the above buffers. It is done. Biological material in an aqueous solution can also be homogenized, ground, or otherwise destroyed to maximize contact between the treatment agent and the biological material.

  In another exemplary embodiment, a cross-linking agent is used to maintain the structural integrity of biological tissue. Structural integrity can be defined as the ability of a tissue to perform its required biological function. Artisans understand that the degree of structural integrity required for an organization to perform its required functions can vary between different types of organizations. Furthermore, the particular application in which the tissue is used may require different levels of structural integrity.

(SCL process)
The method of the invention comprises the step of contacting biological material with a denaturant-free preparation (hereinafter referred to as an SCL preparation) containing a surfactant and a cross-linking agent. Include. Biological materials, crosslinkers, and surfactants useful in the present invention are discussed above and are also useful herein in SCL preparations.

  In the exemplary embodiment, more than one SCL step is performed. In another exemplary embodiment, the SCL process is performed 1 to 5 times. In another exemplary embodiment, the biological material is contacted only once with the SCL preparation.

  Various surfactant concentrations are useful in SCL preparations. In an exemplary embodiment, the tissue is treated with an SCL preparation containing from about 0.1% to about 50% surfactant. In another exemplary embodiment, the tissue is treated with an SCL preparation containing about 0.1% to about 10% surfactant. In another exemplary embodiment, the tissue is treated with an SCL preparation containing greater than 0.6% and less than 50% surfactant. In another exemplary embodiment, the SCL preparation contains about 1.7% to about 25% surfactant. In another exemplary embodiment, the SCL preparation is 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. In another exemplary embodiment, the SCL preparation is 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, Contains 15%, 16%, 17%, 18%, 19% or 20% surfactant.

  As discussed above, various surfactants are useful in the methods (including SCL preparations) of the present invention. In an exemplary embodiment, the surfactant used in the SCL preparation is a nonionic surfactant. In another exemplary embodiment, the nonionic surfactant is Tween 80. In another exemplary embodiment, the SCL preparation is 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 Contains 4%, 2.5%, 2.6%, 2.7%, 2.8%, or 2.9% Tween 80. In another exemplary embodiment, the SCL preparation is 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, Contains 15%, 16%, 17%, 18%, 19% or 20% Tween 80.

  One skilled in the art will recognize that different surfactants can be used in the SCL step of the method of the invention. Determining acceptable surfactants and surfactant levels to be used is well within the ability of one skilled in the art given the guidance of the teachings herein.

  In an exemplary embodiment, the biological tissue is treated with an SCL preparation that contains an aldehyde as a crosslinker. In a related embodiment, the aldehyde is glutaraldehyde. Various glutaraldehyde concentrations can be used in the SCL preparation. In an exemplary embodiment, the preparation contains about 0.1 wt% to about 50 wt% glutaraldehyde. In another exemplary embodiment, the tissue is treated with an SCL preparation containing about 0.2% to about 3% glutaraldehyde.

  Typically, the SCL process is performed at any suitable temperature that is selected to provide optimal calcification reduction, calcification reduction, and / or phospholipid content reduction. In an exemplary embodiment, the SCL process is performed at approximately a single temperature. In another exemplary embodiment, the SCL process is performed at more than one temperature.

  In another exemplary embodiment, the SCL process 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 process is 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. To be implemented.

  Typically, the SCL process is performed for a time selected to provide optimal calcification reduction, calcification reduction or phospholipid content reduction. In an exemplary embodiment, the biological material is contacted with the SCL preparation for about 1 day to about 20 days. In another exemplary embodiment, the SCL process lasts from 5 days to about 15 days.

  In another exemplary embodiment, the SCL process lasts at least about 12 days. In another exemplary embodiment, the SCL process lasts at least about 12 days and the SCL preparation contains at least about 1.7% surfactant.

  In another exemplary embodiment, the SCL process is performed at a pH selected to provide optimal calcification reduction or phospholipid content reduction. In an exemplary embodiment, the pH of the SCL solution is between about 6 and about 8. In another exemplary embodiment, the pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7 .9.

  The SCL process can be performed as a single process. Alternatively, the SCL process can be performed as a series of continuous processes. At the end of each successive step, the SCL preparation is preferably removed from the tissue, which is then contacted with a new fraction of the SCL preparation.

(SCLD process)
The methods of the present invention include contacting a biological material with a preparation (hereinafter referred to as an SCLD preparation) containing a surfactant, a cross-linking agent, and a modifier. . Biological materials, crosslinkers, and surfactants useful in the present invention are discussed above and are also useful herein in SCLD preparations.

  The SCLD process can be performed at different stages of the method of the present invention. In an exemplary embodiment, the SCLD process is performed after the SCL process. In another exemplary embodiment, the SCLD process is performed before the SCL process.

  The SCLD process can also be referred to as a bioburden reduction process. In the 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 referred to as an initial bioburden reduction step (iBReP), and the second SCLD step is It may be referred to as a final bioburden reduction step (fBReP).

  A variety of modifiers are useful in the SCLD process of the present invention. In an exemplary embodiment, the denaturing agent causes a reversible or irreversible loss of a higher order structure other than the primary structure of the protein in the biological material. In another exemplary embodiment, the denaturing agent is a protic solvent useful in removing infectious agents or chemical agents. Protic solvents useful in the practice of the present invention include water, alcohols, carboxylic acids and the like. In another exemplary embodiment, the modifier is an alcohol or other solvent that includes an alcohol. Biological material is treated with virtually any amount of alcohol that provides demanding results.

  Determination of the exact amount of alcohol required for a particular application is well within the ability of those skilled in the art. For example, biological tissue is subjected to one or more SCLD processes and the biological material is recovered. The amount of infectious material or chemical agent removed by this SCLD process is determined. When alcohol stops removing infectious or chemical agents from this tissue and reaches the endpoint, this indicates the amount of alcohol needed to remove a particular agent from the tissue. In an exemplary embodiment utilizing alcohol, 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 methanol, ethyl alcohol, propyl alcohol, butyl alcohol, pentyl alcohol, hexyl alcohol, heptyl alcohol, octyl alcohol, nonyl alcohol, decyl alcohol, long chain alcohols (eg, 1,2-octanediol), and the like. One or more compounds of the group consisting of: In another exemplary embodiment, the method of the invention uses ethanol. In another exemplary embodiment, the alcohol is isopropyl alcohol.

  As discussed above, a variety of crosslinkers are useful in the methods of the present invention (including SCLD preparations). In an exemplary embodiment, the SCLD preparation contains an aldehyde as a crosslinker. In a further exemplary embodiment, formaldehyde is the crosslinker. Various formaldehyde concentrations are useful in SCLD preparations. In an exemplary embodiment, the SCLD preparation contains about 0.5% to about 10% formaldehyde. In another exemplary embodiment, the SCLD preparation contains about 4% formaldehyde.

  The SCLD preparation can use any suitable surfactant. In an exemplary embodiment, the surfactant used in the SCLD preparation is a nonionic surfactant. In another exemplary embodiment, the SCLD preparation contains Tween 80 as a surfactant. In another exemplary embodiment, the SCLD preparation contains about 0.5% to about 5% Tween 80. In another exemplary embodiment, the SCLD preparation contains about 2.1% Tween 80.

  In an exemplary embodiment, the SCLD process is performed at a temperature selected to provide optimal calcification reduction, calcification reduction and / or phospholipid content reduction of the biological material. In an exemplary embodiment, the SCLD process is performed at approximately a single temperature. In another exemplary embodiment, the SCLD process is performed at more than one temperature.

  In another exemplary embodiment, the SCLD process is performed at approximately room temperature. In another exemplary embodiment, heat is applied to the SCLD preparation. In another exemplary embodiment, the SCLD process is performed at a temperature between about 30 ° C. and 55 ° C. In another exemplary embodiment, the SCLD process is performed between about 30 ° C. and about 40 ° C.

  In another exemplary embodiment, two SCLD steps are performed: iBReP step and fBReP step. In another exemplary embodiment, the iBReP step is performed at approximately room temperature and the fBReP step is performed at a temperature above room temperature. In another exemplary embodiment, the fBReP step is performed between about 30 ° C. and about 37 ° C.

  In another exemplary embodiment, each SCLD step is performed for a time selected to provide optimal calcification reduction, calcification reduction, and / or phospholipid content of the biological material. In another exemplary embodiment, the iBReP step is performed for about 1 hour to about 4 hours, and the fBReP step is performed for about 5 hours to about 15 hours.

  In another exemplary embodiment, the SCLD process is performed at a pH selected to provide optimal calcification reduction, mineralization reduction and / or phospholipid content reduction of the biological material. . In another exemplary embodiment, the pH of the SCLD solution is between about 6 and about 8. In another exemplary embodiment, the pH is about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or Maintained at 7.9.

  The SCLD process can be performed as a single process. Alternatively, the extraction can be performed as a series of continuous steps. At the end of each successive step, the SCLD preparation is preferably removed, after which the tissue is contacted with a new fraction of the SCLD preparation.

  In an exemplary embodiment, the SCLD process is performed before the SCL process. In another exemplary embodiment, the SCLD process is performed after the SCL process.

(Contact of biological material with final liquid sterilization solution)
In an exemplary embodiment, the method of the invention further comprises contacting the living tissue with a final liquid sterilization solution. Typically, biological tissue is contacted with the final liquid sterilization solution after completion of the aforementioned SCL and SCLD steps.

  An exemplary final liquid sterilization solution (hereinafter referred to as a “TLS” solution) can sterilize biological material prior to implantation. Sterilizing agents useful in the present invention include crosslinkers, heat, radiation, electron beams, ultraviolet radiation and combinations thereof. In an exemplary embodiment, the TLS solution includes heat and a crosslinker. Exemplary crosslinkers useful in TLS solutions are described above.

  In an exemplary embodiment, the TLS solution is heated to a temperature of about 35 ° C to about 50 ° C. In another exemplary embodiment, the TLS solution is heated to a temperature of about 35 ° C to about 40 ° C.

  In another exemplary embodiment, 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 and 7.5.

  In an exemplary embodiment, the TLS solution is contacted with the biological material for a sufficient time to sterilize the biological material. One skilled in the art recognizes that the time required for sterilization depends on the stringency of the TLS solution. For example, an aldehyde-containing TLS solution that has been heated to about 37 ° C. may require 1-6 days to sterilize biological material, but heating the TLS solution to 50 ° C. will require the time Can be reduced to 25 hours.

  In another exemplary embodiment, the TLS solution comprises about 0.2 wt% to about 2.0 wt% glutaraldehyde.

  In another exemplary embodiment, the final sterilization time and temperature is 1-6 days at 37 ° C. In another particularly preferred embodiment, the final sterilization time and temperature is 1-2 days at 50 ° C. One skilled in the art will recognize that other final sterilization times and temperatures can be adjusted while maintaining the efficiency of the TLS solution.

(Contact between biological material and cross-linking agent)
In an exemplary embodiment, the method of the present invention further comprises contacting the living tissue with a fixative solution comprising a crosslinker in the absence of a denaturing agent. The biological tissue is brought into contact with the fixing solution before the SCL process and the SCLD process, after the SCL process and the SCLD process, or between the SCL process and the SCLD process. Typically, living tissue is contacted with a fixative solution prior to the SCL and SCLD steps.

  Fixing solutions useful in the present invention include cross-linking agents that act on the cross-linking of connective tissue proteins in the tissue. Crosslinkers useful in the present invention are discussed above and are similar to those useful herein in fixing solutions.

  In an exemplary embodiment, the fixing solution includes a cross-linking agent at a concentration of about 0.2% to about 2.0% by weight. In another exemplary embodiment, the cross-linking agent is glutaraldehyde. In another exemplary embodiment, the fixing solution includes glutaraldehyde as a cross-linking agent at a concentration of about 0.2% to about 2.0% by weight.

  In another exemplary embodiment, the fixative solution is contacted with the biological material for about 0.5 hours to about 14 days. In another exemplary embodiment, the fixative solution is contacted with the biological material for about 1-4 days. In another exemplary embodiment, the fixation solution is contacted with the biological material for about 2 days.

  In another exemplary embodiment, the fixing solution further comprises a surfactant. Useful surfactants and surfactant concentrations are described above and are equally applicable to fixing solutions.

  The terms and expressions employed herein are used for descriptive terms, not for limitation, and exclude the features shown and described, or equivalents thereof. It is recognized that such terms and expressions are not intended to be used, and that various modifications are possible within the scope of the claims. Furthermore, any one or more features of any embodiment of the invention may be combined with any one or more features of any other embodiment of the invention without departing from the scope of the invention. Can be. For example, the surfactants and crosslinkers described in the SCL process, and their respective concentrations, are equally applicable to the SCLD process. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

(Example 1)
Example 1 describes a method of mitigating calcification in pericardial tissue, where the pericardial tissue is subjected to treatment with an SCL preparation containing 10% Tween 80 and 0.625% glutaraldehyde.

(1.1 Method)
(1.1.1 Post Tween Process Method)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  Following the preparation of bovine pericardial tissue leaflets, the samples were chemically fixed by immersion in a 0.625% glutaraldehyde solution in aqueous solvent for 30 minutes followed by 7 days of isolation.

  The remaining leaflets were treated with 0.625% glutaraldehyde and 10% Tween 80 in aqueous solvent for 7 days. The leaflets were then subjected to subsequent steps or were implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted and subjected to calcium analysis. Leaflets implanted in rats are referred to hereinafter as “post-Tween process” leaflets in Example 1.

  The remaining leaflets were subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature for 3 consecutive cycles of 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In the fBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature for 4 consecutive cycles of 10 minutes each.

  After the fBReP procedure, the leaflet was subjected to a final liquid sterilization process (TLS). In the TLS procedure, leaflets were treated with 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.2-7.5. The volume of the solution was 100 ml of solution per 3 leaflets. The leaflets were processed for 25-35 hours (C) at 35-40 ° C. (typical minimum and maximum, measured at the internal location of the product).

  Following the TLS procedure, leaflets were implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted, and subjected to calcium analysis, which is referred to herein below in Example 1 as “Tween / TLS process. Call it a leaflet.

(1.1.2 Complete process control method)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  After preparation, leaflets were chemically fixed by treatment with 0.625% glutaraldehyde in aqueous solvent for 14 days. The leaflets were then subjected to subsequent steps or were implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted and subjected to calcium analysis. Leaflets implanted in rats are hereinafter referred to herein as “14-day control” leaflets in Example 1 hereinafter.

  The remaining leaflets were subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature for 3 consecutive cycles of 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In the fBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflet was treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature for 4 consecutive cycles of 10 minutes each.

  After the fBReP procedure, the leaflet was subjected to a final liquid sterilization process (TLS). In the TLS procedure, leaflets were treated with 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.2-7.5. The volume of the solution was 100 ml of solution per 3 leaflets. The leaflets were treated at 35-40 ° C. (typical, lowest and highest, measured at the product internal location) for 25-35 hours.

  After the TLS procedure, leaflets are implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted, and subjected to calcium analysis, hereinafter referred to as “Complete Process Control” leaflets in Example 1.

(1.1.3 Calcium analysis)
After receiving one of the four treatments outlined above, leaflets were implanted subcutaneously into Sprague-Dawley rats for 90 days. The leaflet was removed and prepared for calcium content analysis.

  First, the leaflets were lyophilized, weighed, and then treated with 70% nitric acid at 110 ° C. until fully digested. The digested sample was brought to a final concentration of 3,000 ppm KCl.

  Samples were analyzed for calcium content by atomic absorption spectroscopy (AAS).

(1.2 Results)
FIG. 1 shows the total μg Ca / mg of dry leaflet tissue for continuous treatment using the above four methods; 1) 14-day control; 2) complete process control; 3) post-Tween process; and 4) Tween. / TLS process. The data in FIG. 1 shows a decrease in average calcium content for the Tween / TLS process and the post-Tween process compared to the full process control and the 14 day control.

  FIG. 2 shows a representative example of a diamond plot of the average calcium content reduction data of FIG. The upper and lower ends of the diamonds in FIG. 2 form a 95% confidence interval with respect to the average. The small squares are individual data points. The short horizontal lines near the upper and lower ends of each rhombus are overlapping lines.

  Table 1 shows the calcification mitigation using a post-Tween / TLS process as compared to a full process control leaflet tissue sample containing more than 5 μg Ca / mg mineralized dry leaflet tissue.

  (Table 1)

  Table 1 shows that 7.8% of the tissue from full process control (second group) was calcified, while 0% was calcified in the Tween / TLS process group (fourth group).

(Example 2)
Example 2 describes a method for alleviating calcification in porcine heart valve tissue. Here, porcine heart valve tissue is subjected to treatment with an SCL preparation containing 10% Tween 80 and 0.625% glutaraldehyde.

(2.1 Method)
(2.1.1 Post-Tween process method)
Porcine heart valve tissue leaflets were prepared for chemical fixation by standard techniques.

  After preparation of porcine heart valve tissue leaflets, these samples were chemically fixed by soaking in a solution of 0.625% glutaraldehyde in aqueous solvent for 7 days.

  The remaining leaflet was treated with 0.625% glutaraldehyde and 10% Tween 80 in aqueous solvent for 7 days. The leaflets were then subjected to subsequent steps or transplanted subcutaneously into Sprague-Dawley rats for 90 days and explanted and subjected to calcium analysis. These leaflets implanted in rats are hereinafter referred to in Example 2 as “post-Tween process” leaflets.

  The remaining leaflets were then subjected to an initial bioburden reduction process (iBReP). In this iBreP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. Its volume was more than 100 milliliter (100 ml) solution per 3 leaflets. The leaflets were processed at room temperature for 2 to 2.5 hours.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of this rinse solution was more than 100 milliliters (100 ml) solution per 3 leaflets. This rinsing step was carried out at room temperature over 4 consecutive cycles of 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In this fBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. Its volume was more than 100 milliliter (100 ml) solution per 3 leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of this rinse solution was more than 100 milliliters (100 ml) solution per 3 leaflets. This rinsing step was carried out at room temperature over 4 consecutive cycles of 10 minutes each.

  After this fBReP procedure, the leaflets were subjected to final liquid sterilization (TLS). In this TLS procedure, leaflets were rinsed with 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.2-7.5. The rinse solution volume was 100 ml per three leaflets. Treat the leaflet at 35-40 ° C. (measured at the highest and lowest representative product internal locations) for 25-35 hours, including heating and cooling between 35-40 ° C. did.

  After the TLS procedure, the leaflet is subjected to subsequent steps or implanted subcutaneously over 90 days in Sprague-Dawley rats and explanted and subjected to calcium analysis and thereafter These leaflets are referred to as “Tween / TLS process” leaflets in Example 2.

(2.1.2. Complete process control method)
Porcine heart valve tissue leaflets were prepared for chemical fixation by standard techniques.

  After preparation, the leaflet was chemically fixed by treatment with 0.625% glutaraldehyde in an aqueous solvent for 14 days. The leaflets were then subjected to subsequent steps or implanted subcutaneously in Sprague-Dawley rats for 90 days and explanted and subjected to calcium analysis. These leaflets implanted in rats are hereinafter referred to in Example 2 as “14-day control” leaflets.

  The remaining leaflets were subjected to an initial bioburden reduction process (iBReP). In this iBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. Its volume was more than 100 milliliter (100 ml) solution per 3 leaflets. The leaflets were processed at room temperature for 2 to 2.5 hours.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of this rinse solution was more than 100 milliliters (100 ml) solution per 3 leaflets. This rinsing step was carried out at room temperature for 3 consecutive cycles of 10 minutes each.

  After the iBReP procedure, the leaflet was subjected to a final bioburden reduction process (fBReP). In this fBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. Its volume was more than 100 milliliter (100 ml) solution per 3 leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, these leaflets were treated with a solution of 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of this rinse solution was more than 100 milliliters (100 ml) solution per 3 leaflets. This rinsing step was carried out at room temperature over 4 consecutive cycles of 10 minutes each.

  After this fBReP procedure, the leaflets were subjected to final liquid sterilization (TLS). In this TLS procedure, leaflets were treated with 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.2-7.5. The volume of this rinse solution is 100 ml solution per 3 leaflets. The leaflets were treated at 37.5 ± 2.5 ° C. (measured at the highest and lowest representative product internal locations) for 25-35 hours.

  After the TLS procedure, the leaflet is subjected to subsequent steps or implanted subcutaneously over 90 days in Sprague-Dawley rats and explanted and subjected to calcium analysis and thereafter These leaflets are referred to as “full process control” leaflets in Example 2.

(2.1.3. Calcium analysis)
After performing one of the four procedures outlined above, leaflets were implanted subcutaneously in Sprague-Dawley Rat for 90 days. The leaflet was removed and prepared for calcium content analysis.

  First, the leaflets were lyophilized and weighed and then processed at 110 ° C. with 70% nitric acid until complete digestion. The digested sample was brought to a final concentration of 3,000 ppm for KCl.

  The sample was analyzed for calcium content by atomic absorption analysis (AAS).

(2.2. Results)
FIG. 3 shows total μg Ca / mg of dry leaflet tissue for continuous treatment 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 FIG. 3 shows a decrease in the average calcium content of the Tween / TLS process versus full process control and 14 day control.

  FIG. 4 shows a rhombus plot display of the average calcium content reduction data in FIG. The top and bottom of the diamond in FIG. 2 form a 95% confidence interval for the average. The small squares are individual data points. The short horizontal lines adjacent to the top and bottom of each diamond are overlapping lines.

Table 2 shows calcification mitigation using a post-Tween / TLS process for full process control. A leaflet tissue sample containing tissue exceeding 5 μg Ca / mg dry leaflet tissue is calcified.
(Table 2)

  Table 2 shows that 20% (7/35) of tissue was calcified from full process control (2 groups), while 8.1% (3/37) of Tween / TLS process groups (4 groups) was calcified. Indicates that

(Example 3)
Example 3 describes a method for mitigating calcification in pericardial tissue, wherein the pericardial tissue is treated with an SCL preparation containing 1.2% Tween 80 and 0.625% glutaraldehyde. To serve.

(3.1. Method)
(3.1.1. Control group, leaflet treated with glutaraldehyde for 14 days)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  After preparation of bovine pericardial tissue leaflets, the samples were chemically fixed by immersing them in a 0.625% glutaraldehyde solution in an aqueous solvent for 12-24 hours and then quarantined for 14 days. The sample was then implanted subcutaneously into Sprague-Dawley Rat for 90 days, explanted, and subjected to calcium analysis. These samples are hereinafter referred to as “1 group” leaflets in Example 3.

(3.1.2. Control group, fully processed leaflet)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  After the preparation of bovine pericardial tissue leaflets, the samples were chemically fixed by immersion in a 0.625% glutaraldehyde solution in an aqueous solvent for 12-24 hours and then quarantined for 14 days.

  The leaflet was then subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was more than 100 milliliters (100 ml) of solution per three leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, the leaflet was rinsed in 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of the rinse solution was over 100 milliliters (100 ml) of solution per three leaflets. The rinsing was carried out three times for 10 minutes each at room temperature.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In the fBReP procedure, leaflets were treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was more than 100 milliliters (100 ml) of solution per three leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, the leaflet was rinsed in 0.625% glutaraldehyde in phosphate buffer in an aqueous solvent at pH 7.4. The volume of the rinse solution was over 100 milliliters (100 ml) of solution per three leaflets. The rinsing was performed four times at 10 minutes each at room temperature.

  After the fBReP procedure, the leaflet was subjected to a final liquid sterilization process (TLS). In the TLS procedure, leaflets were treated with a 0.625% glutaraldehyde solution in phosphate buffer in an aqueous solvent at pH 7.2-7.5. The volume of this solution was 100 ml of solution per 3 leaflets. The leaflets were treated at 37.5 ± 2.5 ° C. (typically measured at the coldest and hottest product internal locations) for 25-35 hours.

  After the TLS procedure, the leaflets are implanted subcutaneously into Sprague-Dawley Rat for 90 days, explanted, and subjected to calcium analysis. These samples are hereinafter referred to as “2 group” leaflets in Example 3.

(3.1.3. Test group, leaflets treated with glutaraldehyde for 14 days and glutaraldehyde for 7 days / 1.2% Tween-80)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  After preparing bovine pericardial tissue leaflets, these samples were chemically fixed by immersion in a 0.625% glutaraldehyde solution in aqueous solvent for 12-24 hours and then quarantined on day 7. These leaflets were then treated with 0.625% glutaraldehyde and 1.2% Tween 80 in an aqueous solvent for 7 days.

  These leaflets were then subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde (pH 7.4) in phosphate buffer in an aqueous solvent. The rinse solution volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The rinsing step was carried out at room temperature in 3 consecutive cycles for 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In this fBReP procedure, the leaflet was treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde (pH 7.4) in phosphate buffer in an aqueous solvent. The rinse solution volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The rinsing process was performed at room temperature in four consecutive cycles of 10 minutes each.

  After the fBReP procedure, the leaflet was subjected to a terminal liquid sterilization process (TLS). In this TLS procedure, leaflets were treated with a solution of 0.625% glutaraldehyde (pH 7.2-7.5) in phosphate buffer in an aqueous solvent. The volume of this solution was 100 ml of solution per 3 leaflets. The leaflets were treated at 35-40 ° C. (measured at the coldest and warmest internal locations of a typical product) for 25-35 hours.

  After the TLS procedure, leaflets are implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted, and subjected to calcium analysis, and are referred to herein as “Group 3” leaflets in Example 3.

(3.1.4. Test group, leaflet treated with glutaraldehyde for 1 day, glutaraldehyde / 1.2% Tween 80 for 13 days)
Bovine pericardial tissue leaflets were prepared for chemical fixation using standard techniques.

  After preparing bovine pericardial tissue leaflets, these samples were chemically fixed by immersing them in a 0.625% glutaraldehyde solution in an aqueous solvent for 12-24 hours. These leaflets were then treated with 0.625% glutaraldehyde and 1.2% Tween 80 in aqueous solvent for 13 days.

  These leaflets were then subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde (pH 7.4) in phosphate buffer in an aqueous solvent. The rinse solution volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The rinsing step was carried out at room temperature in 3 consecutive cycles for 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In this fBReP procedure, the leaflet was treated with a formaldehyde-based sterilant solution. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, these leaflets were rinsed with 0.625% glutaraldehyde (pH 7.4) in phosphate buffer in an aqueous solvent. The rinse solution volume was equal to or greater than 100 milliliters (100 ml) of solution per three leaflets. The rinsing process was performed at room temperature in four consecutive cycles of 10 minutes each.

  After the fBReP procedure, the leaflet was subjected to a final liquid sterilization process (TLS). In this TLS procedure, leaflets were treated with a solution of 0.625% glutaraldehyde (pH 7.2-7.5) in phosphate buffer in an aqueous solvent. The volume of this solution was 100 ml of solution per 3 leaflets. The leaflets were treated at 35-40 ° C. (measured at the coldest and warmest internal locations of a typical product) for 25-35 hours.

  After the TLS procedure, leaflets are implanted subcutaneously into Sprague-Dawley rats for 90 days, explanted, and subjected to calcium analysis, and are referred to herein as “Group 4” leaflets in Example 3.

(3.1.3. Calcium analysis)
After performing one of the four treatments outlined above, leaflets were implanted subcutaneously into Sprague-Dawley rats for 90 days. The leaflet was removed and prepared for calcium content analysis.

  The leaflets were first lyophilized and weighed and then treated with 70% nitric acid at 110 ° C. until fully digested. The digested sample was brought to a final concentration of 3,000 ppm KCl.

  The sample was analyzed for calcium content by atomic absorption analysis (AAS).

(3.2. Results)
Table 3 shows the reduction in mineralization using the 7-day Tween 80 test group (Group 3) and the 13-day Tween 80 test group (Group 4) compared to full process control (Group 2). Leaflet tissue samples containing more than 5 μg Ca / mg dry leaflet tissue are calcified.

  (Table 3)

  Table 2 shows that 4.26% (4/94) tissue from full process control (Group 2) is calcified, but 0% from 7 days glutaraldehyde, 7 days Tween 80 test group (Group 3). 0.0% (0/91) of tissue was calcified. In addition, the 2.25% 1-day glutaraldehyde, 13-day Tween 80 test group (Group 4) showed mineralization. Thus, both groups 3 and 4 showed a reduction in calcification compared to the full process control group.

(Example 4)
Example 4 describes a method for reducing phospholipid content in porcine heart valve tissue, wherein porcine heart valve tissue contains 0.6% to 6% Tween 80 and 0.625% glutaraldehyde. It is subjected to treatment with the containing SCL preparation for 7 to 15 days.

(4.1. Method)
(4.1.1. General method)
Porcine heart valve tissue leaflets were prepared for chemical fixation by standard techniques.

  After preparation of the porcine heart valve tissue leaflet, the samples were chemically fixed by immersion for 1-14 days in 0.625% glutaraldehyde solution in aqueous solvent as shown in Table 4.

  The leaflets were then treated with 0.625% glutaraldehyde and 0.6% to 6.0% Tween 80 as shown in Table 4 for 5-15 days.

  The leaflet was then subjected to an initial bioburden reduction process (iBReP). In the iBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80, and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflet was treated at room temperature for 2-2.5 hours.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature in 3 consecutive cycles of 10 minutes each.

  After the iBReP procedure, the leaflets were subjected to a final bioburden reduction process (fBReP). In the fBReP procedure, leaflets were treated with a solution of formaldehyde-based sterilant. This solution contained 4% formaldehyde, 22% ethanol, 1.2% Tween 80 and 11.3 mM phosphate buffer. The volume was equal to or more than 100 ml of solution per 3 leaflets. The leaflets were treated at 30-37 ° C. for 10 hours ± 30 minutes.

  After treatment, the leaflets were rinsed in 0.625% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.4. The rinse solution volume was equal to or greater than 100 ml solution per three leaflets. The rinse was performed at room temperature in four consecutive cycles of 10 minutes each.

  After the fBReP procedure, the leaflet was subjected to a final liquid sterilization process (TLS). In the TLS procedure, leaflets were treated with 0.516-0.656% glutaraldehyde phosphate buffer in an aqueous solvent at a pH of 7.2-7.5. The volume of the solution was 100 ml of solution per 3 leaflets. The leaflets were treated for 25-35 hours at 35-40 ° C. (typical minimum and maximum, measured at the internal location of the product).

(4.1.2. Phospholipid analysis)
After receiving one of the four treatments outlined above, leaflets were implanted subcutaneously into Sprague-Dawley rats for 90 days. Leaflets were explanted and prepared for phospholipid content analysis.

  The leaflet was frozen in liquid nitrogen, ground and dissolved in an extraction solvent containing chloroform and methanol. Following the addition of saline, precipitated proteins and most tissues were removed by centrifugation. The extracted liquid was then recovered 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.

(4.2. Results)
Table 4 shows the decrease in phospholipid content in the method using the SCL step compared to the method without the SCL step.

  (Table 4)

  +++ indicates less than 0.200 μg PL / mg dry tissue.

  ++ indicates greater than 0.200 μg PL / mg dry tissue and less than 0.400 μg PL / mg dry tissue.

  + Indicates greater than 0.400 μg PL / mg dry tissue and less than 1.000 μg PL / mg dry tissue.

  --- indicates greater than 1.00 μg PL / mg dry tissue.

FIG. 1 shows data from an experiment to assess 90 days of rat subcutaneous pericardial leaflet mineralization (μg Ca / mg dry weight) as described in Example 1 below. FIG. 2 shows data from an experiment to assess 90 days of rat subcutaneous pericardial leaflet calcification (μg Ca / mg dry weight) as described in Example 1 below. FIG. 3 shows data from an experiment to assess 90 days of subcutaneous pork leaflet calcification (μg Ca / mg dry weight) as described in Example 2 below. FIG. 4 shows data from an experiment to evaluate 90 days of subcutaneous pork leaflet calcification (μg Ca / mg dry weight) as described in Example 2 below.

Claims (24)

A method of processing a biological material, the method comprising:
(A) contacting the biological material with a preparation comprising a surfactant and a crosslinking agent in the absence of the modifying agent; and (b) a preparation comprising the surfactant, the crosslinking agent and the modifying agent. Contacting the biological material;
Including the method. The method of claim 1, wherein when the method is implanted into a host organism, the method results in reduced calcification in the biological material as compared to the same method that does not include step (a). The method of claim 2, wherein the reduction of calcification results in the elimination of calcification in the biological material. The method of claim 1, wherein the method results in a reduction in phospholipid content in the biological material when implanted in a host organism as compared to the same method that does not include step (a). The method according to claim 1, wherein the step (a) is performed before the step (b). The method of claim 1, wherein the method further comprises contacting the biological material with a final liquid sterilized solution after completion of steps (a) and (b). The method further comprises contacting the biological material with a solution containing a cross-linking agent in the absence of a denaturant and in the absence of a surfactant prior to step (a). The method of claim 6. The method of claim 1, wherein the biological material is bioprosthetic tissue. The method of claim 8, wherein the bioprosthetic tissue is incorporated into a bioprosthesis. The method of claim 6, wherein the final liquid sterilization solution comprises a cross-linking agent and is heated to a temperature in the range of 35-55 ° C. The method according to claim 1, wherein the crosslinking agent of the preparation used in steps (a) and (b) is: aldehyde, diisocyanate, carbodiimide, polyepoxy compound, difunctional maleimide compound, difunctional Independently selected from the group consisting of: a functional N-hydroxysuccinimide ester compound, a bifunctional imide ester compound, a bifunctional pyridylthio compound, a bifunctional vinyl sulfone compound, a photoactivatable crosslinking agent, or combinations thereof. Method. The method according to claim 11, wherein the cross-linking agent of the preparation used in step (a) is glutaraldehyde. The method of claim 1, wherein the cross-linking agent of the preparation used in step (b) is a member selected from the group consisting of: formaldehyde, glutaraldehyde, or combinations thereof. The method of claim 1, wherein the modifier of the preparation used in step (b) is a protic solvent. 15. A method according to claim 14, wherein the modifying agent of the preparation used in step (b) is an alcohol. 16. A method according to claim 15, wherein the denaturing agent of the preparation used in step (b) is ethanol. The method according to claim 1, wherein the surfactant of the preparation used in step (a) is selected from the group consisting of nonionic surfactants. 18. A method according to claim 17, wherein the surfactant of the preparation used in step (a) is Tween 80. 19. The method of claim 18, wherein the concentration of Tween 80 is between 0.7% and 15%. 20. The method of claim 19, wherein the concentration of Tween 80 is selected between 1.6% and 11%. A method of processing a biological material, the method comprising:
(A) a first step of contacting the biological material with a preparation comprising a surfactant, a crosslinker and a denaturant; and (b) after step (a), comprising a surfactant and a crosslinker. Contacting the preparation with the biological material;
(C) after step (b), contacting the biological material with a preparation comprising a surfactant, a crosslinking agent and a denaturing agent; and (d) after step (c), a final liquid sterilization solution. Contacting the biological material with the biological material,
Including the method. 24. The method of claim 21, wherein when the method is implanted in a host organism, the method results in reduced calcification in the biological material as compared to the same method that does not include step (b). 24. The method of claim 21, wherein the method results in a reduction in phospholipid content in the biological material when implanted in a host organism as compared to the same method that does not include step (b). A biological material that is resistant to calcification, including:
(A) contacting the biological material with a preparation comprising a surfactant and a crosslinking agent in the absence of the modifying agent; and (b) a preparation comprising the surfactant, the crosslinking agent and the modifying agent. Contacting the biological material;
A biological material produced by a method of processing the biological material.

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