Compositions and methods for tissue sterilization

Abstract

The present invention provides a sterilization system, composition, and method for sterilizing biological specimens using nitric oxide generated in the solution in which the specimens are placed. In some embodiments, the nitric oxide is released from a nitric oxide precursor material that can be formed in situ from a suitable reagent. Advantageously, the sterilization can be carried out at ambient temperature and pressure, using nitric oxide as the sterilizing agent, thereby preserving the biochemical and / or biomechanical properties of the sterilized tissue. Furthermore, this sterilization process does not require specialized equipment, and the sterilization solution can be disposed of after use without adversely impacting the environment.

Description

Detailed description of the invention

[0001]

[0001] This application claims priority to our concurrently pending U.S. Provisional Patent Application No. 63 / 447,811, filed on 23 February 2023 and incorporated herein by reference.

[0002] [Field of Invention]

[0002] The field of the present invention relates to the sterilization of biological materials, and in particular to the sterilization of biological donor tissue for storage and implantation.

[0003] [Background of the Invention]

[0003] The background description contains information that may be helpful in understanding the present invention. Not any information provided herein constitutes prior art or is related to the claimed invention, nor is any publication specifically or implicitly referenced considered prior art.

[0004]

[0004] All publications and patent applications herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference. If there is any inconsistency in the definition or use of a term in an incorporated reference or which contradicts the definition of a term provided herein, the definition provided herein shall apply, and the definition of the term in the reference shall not.

[0005]

[0005] The use of human donor allografts to repair damaged tissue has resulted in significant functional improvements and improved quality of life in many individuals receiving such allografts. Unfortunately, although the risks of tissue transplantation are relatively low, disease transmission remains a major concern, and therefore, sterilization of allografts is necessary to reduce the possibility of viral or bacterial transmission from the donor to the patient. Numerous sterilization methods are known in the art, but many of these are unsuitable for allografts due to the harsh physical and / or chemical effects they have on them. In fact, maintaining the biomechanical properties of the tendon is crucial for successful functional recovery, especially when the allograft is a tendon.

[0006]

[0006] In other reported methods, tendons are sterilized by radiation using electron beam or gamma irradiation, or by using various chemicals such as supercritical carbon dioxide, ethylene oxide, hydrogen peroxide, chlorhexidine, or by using a multi-stage process (BioCleanse) that uses various chemical solutions and various pressure cycles. Unfortunately, all currently known methods result in some degree of damage or alteration to the tissue after treatment, and in some cases the tissue is not sufficiently sterilized, leaving a risk of disease transmission to the recipient (see, for example, BMC Musculoskeletal Disorders (2021) 22:404). Thus, sterilization of allografts requires a compromise between the effectiveness of sterilization and the rigor of the sterilization process, which frequently affects the biochemical and biomechanical properties. Furthermore, many of the known processes produce a considerable amount of waste that requires treatment before being disposed of in the sewage system environment.

[0007]

[0007] In another example of tissue sterilization, nitric oxide radicals are released from a film placed in the mouth of a subject. The film is then irradiated with light to break down the nitroprusside in the film, generating nitric oxide radicals, thereby locally treating gingival disease caused by microorganisms, as described in U.S. Patent Application Publication 2004 / 02652. Although conceptually relatively simple, the ingestion of materials used can be a problem. Furthermore, lighting is required to generate nitric oxide in situ, which is not always feasible, and even undesirable.

[0008]

[0008] Thus, although various compositions and methods for sterilizing biological tissues are known in the art, all or most of them have some drawbacks. Therefore, improved compositions and methods are still needed that sterilize biological tissues by gentle and effective means that preserve their biochemical and biomechanical properties and produce an environmentally friendly waste flow.

[0009] [Overview of the prefecture]

[0009] The subject matter of the present invention relates to various compositions, systems, and methods for sterilizing various biological materials that preserve the biochemical and / or biomechanical properties of the biological materials and produce environmentally friendly waste flows. The sterilization procedures presented herein are advantageous because they do not require special safety devices and can be carried out at ambient temperature and pressure.

[0010]

[0010] In one aspect of the subject matter of the present invention, the inventors provide a method for sterilizing biological tissue, wherein the number of bacteria in and / or on the tissue is at least 10 4 The proposed method includes the step of placing the tissue in a sterile solution for a time sufficient to reduce the number of bacteria to one-tenth, wherein the solution contains a nitric oxide precursor material that decomposes and releases a sufficient amount of nitric oxide to reduce the number of bacteria, and the nitric oxide precursor material is optionally generated in situ by reacting a first reagent and a second reagent in the solution.

[0011]

[0011] In preferred embodiments, the tissue is human donor tissue for transplantation (e.g., tendon, cornea, bone, or skin), and / or the sterilization solution is an aqueous solution. Optionally, the aqueous solution may further contain osmotic molar concentration adjusters, nutrients, and / or cleaning agents. In further embodiments, the sterilization time is 60 to 300 minutes, or 300 to 1,200 minutes, or 1,200 to 2,800 minutes (and even longer), and generally more preferred (but not required) is that the tissue is sterilized in the solution at a temperature of 4°C to 37°C, and / or the tissue is sterilized in the solution at a pressure of 900 to 1,100 mbar. Most typically, the intended method aims to reduce the bacterial count to at least 10 6 1 / 1, or at least 10 7 1 / 1, in some embodiments at least 10 8 It makes it possible to reduce it to one-tenth.

[0012]

[0012] In exemplary embodiments, the tissue is a tendon, and it is intended that the mechanical tissue parameters of the tendon after sterilization (e.g., fracture load, ultimate tensile strength, and Young's modulus) remain substantially unchanged (e.g., change of 10% or less).

[0013]

[0013] In yet another embodiment, the nitric oxide precursor material comprises a nitrosothiol and can be added to the sterilization solution before the tissue is placed in the sterilization solution, or the first and second reagents are a thiol-containing compound and a nitrosated compound that react in solution to form a nitrosothiol, respectively. Preferably, but not essential, the nitrosothiol, or the thiol-containing compound and the nitrosated compound are provided in a dry formulation (e.g., tablets or powder).

[0014]

[0014] Accordingly, the inventors intend a method for sterilizing tendon tissue while maintaining the mechanical properties of the tendon tissue (e.g., fracture load, ultimate tensile strength, and / or Young's modulus). Such a method reduces the number of bacteria in and on the tissue to at least 10 4A step of placing tendon tissue in a sterile solution for a time sufficient to reduce the number of bacteria to one-tenth, wherein the solution contains a nitric oxide precursor material that decomposes and releases a sufficient amount of nitric oxide to reduce the number of bacteria, and the nitric oxide precursor material is optionally produced in situ by reacting first and second reagents in the solution.

[0015]

[0015] In such a method, the sterilization solution is an aqueous solution, where the nitric oxide precursor material contains a nitrosothiol, and the first and second reagents are generally preferably a thiol-containing compound and a nitrosated compound, respectively. Furthermore, it is preferable that the tissue is sterilized in the solution at a temperature of 4°C to 37°C and / or that the tissue is sterilized in the solution at a pressure of 900 to 1,100 mbar. As recognized, the method intended to reduce the bacterial count to at least 10 6 It is suitable for reducing to one-tenth.

[0016]

[0016] Accordingly, the inventors intend to provide an intermediate sterilization composition comprising biological tissue immersed in a sterilization solution. In such a composition, the sterilization solution contains a nitric oxide precursor material that decomposes in the solution to release a sufficient amount of nitric oxide to reduce the number of bacteria in and / or on the tissue. Most typically, the nitric oxide precursor material reduces the number of bacteria in and / or on the tissue to at least 10 after the tissue has been exposed to nitric oxide for 6 hours or less. 4 It has a chemical composition that releases an amount of nitric oxide that reduces the amount by 1 / 2.

[0017]

[0017] In preferred embodiments, the mechanical properties of the tissue remain substantially unchanged (e.g., changes in the fracture load, ultimate tensile strength, and / or Young's modulus of the tendon after sterilization are 10% or less), and the sterilization solution has a temperature of 4 to 37°C and a pressure of 900 to 1,100 mbar.

[0018]

[0018] Various objects, features, aspects, and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which similar reference numerals indicate similar components.

Brief Description of the Drawings

[0019] [Figure 1] A photograph showing exemplary results of direct inoculation onto a human tendon followed by sterilization, along with a control. [Figure 2A] A photograph showing exemplary results of preloading a human tendon for 24 hours followed by sterilization of the bioburden, along with a control. [Figure 2B] A photograph showing exemplary results of preloading a human tendon for 24 hours followed by sterilization of the bioburden, along with a control. [Figure 3] A figure showing a photograph of an experimental apparatus for determining the mechanical properties of tendon materials tested using an exemplary tissue sterilization solution. [Figure 4] A graph showing a comparison of exemplary stress-strain curves of human tendons tested using an exemplary tissue sterilization solution and sterile saline respectively. [Figure 5] A graph showing a comparison of exemplary stress change curves over time of human tendons tested using an exemplary tissue sterilization solution and sterile saline respectively.

[0020] [Detailed Description]

[0024] The inventors have discovered various compositions and methods for sterilizing tissue that can be carried out using ordinary reagents that generate sufficient amounts of nitric oxide in situ to sterilize tissue in a relatively short time (e.g., 2 - ~6 hours, or 6 hours and 24 hours) after contact with the tissue under ambient conditions (e.g., temperature 20 - 25°C, atmospheric pressure, and relative humidity of 20 - 80%). Advantageously, the sterilization process is carried out with a solution that can be adjusted according to the specific needs of the tissue, and the solution can be replaced with a transport or storage solution after sterilization is complete.

[0021]

[0025] Furthermore, it should be noted that the solutions presented herein can sterilize excised allografts, decellularized tissues, cellularized matrices, various soft and hard tissues, membranes, and various biological tissues, including cells such as nerves, bone marrow cells, and T cells, without damaging or altering the biochemical and / or biomechanical properties of the tissues. It should also be recognized that the solutions presented herein are environmentally friendly, do not require special personal protective equipment for use, and can be disposed of without additional treatment.

[0022]

[0026] In one embodiment, the sterilization solution contains a nitrosothiol that decomposes at a rate sufficient to produce an effective amount of nitric oxide to generate the sterilization solution. As is readily apparent, the nitrosothiol may be added to the solution (as a compound or in conjunction with a carrier) or generated in situ from a precursor material. Therefore, it should be recognized that nitric oxide is formed in situ from a precursor compound in the solution, and that the nitric oxide exerts a sterilizing effect on the tissue while the tissue is present in the solution. Advantageously, the solution can be adapted to specific tissues and therefore adjusted to appropriate osmotic molar concentrations, nutrients, and / or salt content, and may further contain one or more pharmaceutically active agents to maintain or preserve a specific tissue state.

[0023]

[0027] For example, nitrosothiols may be S-nitrosoglutathione or nitrosocysteine, and can typically be formed in situ by the reaction of glutathione or cysteine ​​with sodium nitrite in aqueous solution. Most typically, nitrosothiols or other nitric oxide precursors are present in the solution in amounts of 1–20 mM, 20–50 mM, 50–100 mM, or even greater. As recognized, the sterile solution thus prepared may be isotonic (or adjusted to isotonicity using a suitable salt or other isotonic agent), and therefore may be 0.9% physiological saline. Donor tissue is then placed in the sterile solution and incubated for 2–6 hours, typically at room temperature and atmospheric pressure, to achieve the desired degree of sterility (e.g., SAL is 10%). -6The following conditions are met: After sterilization, if desired, the donor tissue is washed with sterile saline (or other suitable solution) and transported to the storage or use location.

[0024]

[0028] Regarding suitable nitric oxide precursor materials, any material should be considered suitable as long as it produces a sterile amount of nitric oxide over a predetermined period of time. Therefore, intended nitric oxide precursor materials include reaction products of thiol-containing compounds and nitrosated compounds (which may be formed in situ in the sterile solution or prepared beforehand), reaction products of thiolactones, primary amines, and nitrosated compounds (which may be formed in situ in the sterile solution or prepared beforehand), reaction products of thiol-containing alcohols, silanes, and nitrosated compounds (which may be formed in situ in the sterile solution or prepared beforehand), and / or modified polymers containing nitric oxide precursor materials. As recognized, the nitric oxide precursor material decomposes to produce nitric oxide, which then acts as a sterilizing agent.

[0025]

[0029] Furthermore, in at least some embodiments, the generation of nitric oxide by the decomposition of the nitric oxide precursor material can be controlled by controlling specific reaction conditions (e.g., supply of thermal energy or acoustic energy, illumination, or RF irradiation, change in pH, etc.). Further, it should be recognized that the decomposition rate can be at least partially determined by the type of nitric oxide precursor material. For example, a nitric oxide precursor material having a primary nitroso thiol group decomposes faster than one having a secondary nitroso thiol group. Thus, the release of nitric oxide can be tailored to a specific profile using primary and / or secondary nitroso thiols, along with modification or adjustment of appropriate environmental parameters. As will be readily appreciated, the nitric oxide precursor material may be provided as a final compound (typically a dry formulation) that is later typically placed in an aqueous solution, or the nitric oxide precursor material may be formed in situ, typically in an aqueous solution, by the reaction of the components as described above.

[0026]

[0030] For example, when the nitric oxide precursor material is formed in situ from a thiol-containing compound and a nitrosating compound, exemplary thiol-containing compounds include 1,2-ethanedithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-propanedithiol, 2-hydroxypropanethiol, 1-mercapto-2-propanol, dithioerythritol, and dithiothreitol. Other exemplary thiol-containing compounds include alpha-lipoic acid, methanethiol (CH3SH [m-mercaptan]), ethanethiol (C2H5SH [e-mercaptan]), 1-propanethiol (C3H7SH [n-P mercaptan]), 2-propanethiol (CH3CH(SH)CH3 [2C3 mercaptan]), butanethiol (C4H9SH ([n-butyl mercaptan])) tert-butyl mercaptan (C(CH3)3SH [t-butyl mercaptan]), pentanethiol (C5H 11SH (pentyl mercaptan), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 2-mercaptonidole, transglutaminase, (11-mercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functionalized gold nanoparticles, 1,1',4',1"-terphenyl-4-thiol, 1,11-undecanedithiol, 1,1 6-Hexadecanedithiol, 1,2-Ethanedithiol, 1,3-Propanedithiol, 1,4-Benzenedimethanethiol, 1,4-Butanedithiol, 1,4-Butanedithiol diacetate, 1,5-Pentanedithiol, 1,6-Hexanedithiol, 1,8-Octanedithiol, 1,9-Nonanedithiol, Adamantanethiol, 1-Butanethiol, 1-Decanethiol, 1-Dodecanethiol, 1-Heptanethiol, 1-Heptanethiol Plum, 1-Hexadecanedithiol, 1-Hexanethiol, 1-Mercap To-(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanthioll, 1-octadecanethiol, 1-octanthiol, 1-octanthiol, 1-pentadecanethiol, 1-pentanethiol, 1-propanthioll, 1-tetradecanethiol, 1-tetradecanethiol plum, 1-undecanethiol, 11-(1H-pyrrole-1-yl)undecane-1-thiol, 11-amino-1-undecanethiol hydrochloride, 11-bromo -1-Undecanethiol, 11-Mercapto-1-Undecanol, 11-Mercapto-1-Undecanol, 11-Mercapto-Undecanoic Acid, 11-Mercapto-Undecanoic Acid, 11-Mercapto-Undecyl Trifluoroacetate, 11-Mercapto-Undecyl Phosphate, 12-Mercapto-Dodecanoic Acid, 12-Mercapto-Dodecanoic Acid, 15-Mercapopentadecanoic Acid, 16-Mercaptohexadecanoic Acid, 16-Mercaptohexadecanoic Acid, 1H,1H,2H,2H-Perfluoroorodecanethiol, 2,2'-(Ethylenedioxy)Diethanethiol, 2,3-Butanedithiol, 2-Butanethiol, 2-Ethylhexanthiol, 2-Methyl-1-propanthiol, 2-Methyl-2-propanthiol, 2-Phenylethanethiol, 3,3,4,4,5,5,6,6,6-Nonafluoro-1-Hexanethiol Plum, 3-(Dimethoxymethylsilyl)-1-propanthiol, 3-Chloro-1-propanthiol, 3-Mercapto-1-propanol, 3-Mercapto-2-Butanol, 3-Mercapto-N-Nonylpropionamide, 3-Mercapto Propionic acid, 3-mercaptopropyl functionalized silica gel, 3-methyl-1-butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-butanol, 6-(ferrocenyl)hexanthiolate, 6-mercapto-1-hexanol, 6-mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-mercapto -1-nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate, copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol-functionalized silver nanoparticles, dodecanethiol-functionalized gold nanoparticles, dodecanethiol-functionalized silver nanoparticles, hexa(ethylene glycol) mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-mercaptopropionate, octanthiol-functionalized gold nanoparticles, PEG dithiol, Examples include S-(11-bromoundecyl)thioacetate, S-(4-cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11-mercaptoundecyl ether, trimethylolpropanetris(3-mercaptopropionate), [11-(methylcarbonylthio)undecyl]tetra(ethylene glycol), m-carborane-9-thiol, ρ-terphenyl-4,4”-dithiol, tert-dodecyl mercaptan, and / or tert-nonyl mercaptan.

[0027]

[0031] In certain embodiments, thiol-containing compounds include cysteine ​​or its derivatives, thiol-derivative polymers or fillers, or combinations thereof. In embodiments utilizing cysteine ​​or its derivatives, cysteine ​​or its derivatives may include cysteine, glutathione, acetylcysteine, penicillamine, acetylpenicillamine, S-nitroso-n-acetylpenicillamine, bucillamine, or combinations thereof. It should be recognized that thiol-containing compounds may be included as part of a peptide or other macromolecule, provided they are compatible with the components of the multicomponent composition. In embodiments utilizing cysteine ​​or its derivatives as part of a peptide, the peptide may contain any combination of amino acids, provided that the peptide contains cysteine ​​or its derivatives as at least one component of the peptide. A non-limiting example of suitable cysteine ​​or its derivatives is described in the academic paper titled "S-Nitrosothiol Detection via Amperometric Nitric Oxide Sensor with Surface Modified Hydrogel Layer Containing Immobilized Organoselenium Catalyst," cited in Langmuir 2006, 22, 25, 10830-10836, which is incorporated herein by reference in its entirety. Further considerations, aspects, and embodiments are described in our concurrently pending international patent application publication No. 2023 / 219811, which is incorporated herein by reference.

[0028]

[0032] In another example, when the nitric oxide precursor material is formed from a thiolactone, a primary amine, and a nitrosated compound, the thiolactones of particular interest include those having an α-acetothiolactone group, a β-propiothiolactone group, a γ-butyrothiolactone group, a δ-valerothiolactone group, an ε-caprothiolactone group, a ζ-enanthothiolactone group, an η-caprylothiolactone group, and a θ-pelargothiolactone group. In some embodiments, the thiolactone is an amine-containing thiolactone such as thietanone (e.g., N-(2,2-dimethyl-4-oxo-3-thietanyl)acetamide).

[0029]

[0033] In various embodiments, suitable primary amines in conjunction with the teachings presented herein include cysteine ​​or its derivatives, lysine or its derivatives, butylamine or its derivatives, or combinations thereof. In other embodiments described above, the primary amine contains a thiol functional group. In exemplary embodiments, cysteine ​​or its derivatives include cysteine, glutathione, acetylcysteine, penicillamine, acetylpenicillamine, S-nitroso-n-acetylpenicillamine, bucillamine, or combinations thereof. As readily recognizable, this thiol functional group also participates in nitrosation reactions and may therefore accrue additional nitrosyl groups for decomposition to nitric oxide. Further discussion, aspects, and embodiments are described in our concurrently pending international patent application PCT / US23 / 80533, filed November 20, 2023, and incorporated herein by reference. As readily recognizable, secondary and tertiary amine compounds are also expressly intended for use herein.

[0030]

[0034] In yet another example, when the nitric oxide precursor material is formed from a thiol-containing alcohol, a silane, and a nitrosated compound, the thiol-containing alcohol may be 3-mercapto-3-methyl-1-butanol, 2-mercaptoethanol, 3-mercaptohexanol, 4-mercapto-3-methyl-2-butanol, 3-mercapto-2-methyl-1-butanol, 3-mercapto-2-methyl-1-pentanol, (+ / -)-4-mercapto-4-methyl-2-pentanol, 2-mercapto-2-methyl-1-pentanol, 4-mercapto-4-methyl-pentano-2-ol, 3-mercaptohexane-1-ol, or a combination thereof, and the silane may be tetramethoxysilane, tetraethoxysilane, etc. Further considerations, aspects, and embodiments are described in our concurrently pending international patent application PCT / US23 / 79150, filed on 8 November 2023 and incorporated herein by reference.

[0031]

[0035] The intended nitrosated compounds suitable for use with the above reagents include those that can act as nitroso group donors and are therefore compounds of the formula NOX [wherein X is an organic or inorganic anion, or an OR2 group (wherein R2 is an organic group)]. In such compounds, X may be an organic anion derived from a carboxylic acid, for example, an alkane carboxylic acid containing 2 to 7 carbon atoms (for example, acetyl nitrite and propionyl nitrite are examples of this type of nitrosating agent). When X is an inorganic anion, X may be derived from, for example, mineral acids, such as halide ions such as chloride ions or bromide ions, or sulfate ions, or Lewis acids, such as borofluoride ions. Other inorganic anions include hydroxides and sulfonates. Therefore, examples of this type of nitrosated compounds include nitrosyl chloride, nitrosyl sulfate, nitrosyl borofluoride, nitrite, and Flemy salt (potassium nitrosyldisulfonate). If X is a formula OR2 group, the organic group R2 may be, for example, a lower alkyl group containing 1 to 9 carbon atoms, such as ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or isopentyl.

[0032]

[0036] In certain embodiments, the nitrosating compound includes a nitrite. The nitrite may include sodium nitrite, calcium nitrite, potassium nitrite, tetrabutylammonium nitrite, dicyclohexylammonium nitrite, butyl nitrite, isobutyl nitrite, t-butyl nitrite, amyl nitrite, pentyl nitrite, nitrites, ion-pair nitrites, silver nitrite, zinc nitrite, iron nitrite, copper nitrite, transition metal nitrite compounds, or combinations thereof. In yet another embodiment, nitric oxide gas can be used as the nitrosating agent.

[0033]

[0037] In certain embodiments, the reaction to produce a nitric oxide precursor material from a nitrosated compound(s) is typically carried out in an aqueous solvent such as water or a mixture of water with methanol or ethanol. However, depending on the type of reactants, the solvent may also include organic solvents such as tetrahydrofuran. Other non-limiting examples of suitable solvents include various aromatic compounds, aliphatic compounds, ketones, e.g., methyl ethyl ketone, isobutyl ketone, ethyl amyl ketone, acetone, etc., alcohols, e.g., methanol, ethanol, n-butanol, isopropanol, etc., esters, e.g., ethyl acetate, etc., glycols, e.g., ethylene glycol, propylene glycol, etc., ethers, e.g., tetrahydrofuran, ethylene glycol monobutyl ether, etc., or combinations thereof. Therefore, it should be recognized that when the nitric oxide precursor material is prepared in situ in a sterilization solution, a physiologically acceptable solvent or solvent mixture (i.e., one that does not denature tissue or cells and / or alter the mechanical properties of tissue) is particularly preferred, whereas there are no limitations when the nitric oxide precursor material is produced before being combined with the sterilization solution.

[0034]

[0038] In yet another example, if the nitric oxide precursor material is a polymer modified to contain a nitric oxide precursor, the polymer may, at least temporarily, be a carrier that holds the intended nitric oxide precursor material or the reagent used to form the nitric oxide precursor material, or the polymer may be chemically modified to contain a covalently bonded nitric oxide precursor material. As is readily apparent, such polymers may be soluble or insoluble, swellable or non-swellable, and / or organic or inorganic polymers. It should be recognized that all precursor materials and reagents forming the precursor material may be blended into a polymer, solution phase, or powder and used for nitric oxide production to create a variety of NO donors / producers by blending the nitric oxide precursor into a matrix (a polymer, or powder, or one embedded in or on a solid carrier), or to create a solution that produces NO from the matrix / solution phase in a controlled and predictable manner. Exemplary suitable polymer materials and their manufacture are described in our concurrently pending International Patent Application Publication No. 2023 / 205125, which is incorporated herein by reference.

[0035]

[0039] Regarding the appropriate amount of nitric oxide release, it is generally intended that the nitric oxide precursor material be present in the solution in amounts of at least 0.1 mM, at least 0.5 mM, at least 1.0 mM, at least 3.0 mM, at least 6.0 mM, at least 10 mM, at least 25 mM, at least 50 mM, at least 100 mM, or more, so that it releases a sterile amount of nitric oxide over time (e.g., at least 5 minutes, or at least 15 minutes, or at least 30 minutes, or at least 45 minutes, or at least 45 minutes, or at least 6 hours, or longer). Therefore, appropriate amounts of nitric oxide precursor material range from 0.1 to 1.0 mM, or 1.0 to 10 mM, or 10 to 25 mM, or 25 to 50 mM, or 50 to 100 mM. As is readily apparent, when adding reagents to a solution to produce nitric oxide, the amount of each reagent is selected so as to yield the desired amount of nitric oxide precursor material.

[0036]

[0040] Therefore, it should be recognized that a sterilization solution can be produced at the time of use by mixing solid or liquid reactants and then adding the reactants to the sterilization solution, or by adding solid or liquid reactants to the sterilization solution. Similarly, a sterilization solution can also be produced at the time of use by mixing a nitric oxide precursor material with the solution. In further embodiments, the sterilization solution already contains a nitric oxide precursor material, or a reagent that produces a nitric oxide precursor material, and the release of nitric oxide is initiated by irradiation, a change in pH, illumination, etc. Alternatively, the nitric oxide in the intended solution is preferably formed in situ in the solution at the time of use. As mentioned above, a suitable solution is typically an aqueous solution, but non-aqueous solutions are not excluded herein. Furthermore, the sterilization solution may include additional components that can provide specific functions (e.g., adjustment of osmotic molar concentration and / or pH, addition of nutrients, antifungal activity, protease inhibition, addition of cleaning agents, etc.).

[0037]

[0041] As is readily apparent, nitric oxide precursor materials, or components for generating nitric oxide precursor materials in situ, can be provided in various forms, particularly suitable of which are liquids in which the components are usually physically separate liquids that can be mixed (either by themselves or in solution) before use. On the other hand, if the nitric oxide precursor material is solid (or combined with a solid), such solid is intended to be formulated as tablets or powders. Similarly, if the components for generating nitric oxide precursor materials in situ are solid, the solids may be provided separately, or more preferably, in combination in a dry form (e.g., tablets or powders). When the solid form is added to a solution, the nitric oxide precursor is released, or the components react to form the nitric oxide precursor material.

[0038]

[0042] Regardless of the type of formulation and reagents used, the sterile solution is intended to be prepared within a relatively short time of use (i.e., contact time with cells or tissues). For example, the time from preparation to use of the sterile solution is typically less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 5 minutes. Similarly, the contact period between cells or tissues and the sterile solution is generally preferably 15 seconds to 1 minute, or 1 to 15 minutes, or 15 to 60 minutes, or 60 to 180 minutes, or 2 to 6 hours, and may be longer in some cases (e.g., if the solution is also used for temporary storage of 12 hours to 3 days). However, significantly longer times are also explicitly intended herein, including 6 to 12 hours, or 12 to 18 hours, or 18 to 24 hours, or 24 to 48 hours. Therefore, sterilization can be carried out as a relatively rapid process without subsequent storage, or as a slower process that may include additional storage time in the same solution without further sterilization (e.g., when it is no longer needed or when no more nitric oxide precursor material is available).

[0039]

[0043] Therefore, suitable timeframes for sterilizing cells or tissues are at least 1 minute, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, and typically less than 48 hours, less than 24 hours, less than 12 hours, less than 10 hours, less than 8 hours, and even less than 6 hours. Such timeframes generally allow for a bacterial count of at least 10% in and / or on the tissue. 4 1 / 1, or at least 10 5 1 / 1, or at least 10 6 1 / 1, or at least 10 7 1 / 1, or at least 10 8 It is suitable for achieving sterilization that reduces the amount by 1 / 2. Another way to look at it is that the intended sterilization solution is (typically within the above time frame) 10 -4 The following SAL, 10 -5 The following SAL, or 10 -6 The following SAL can be obtained.

[0040]

[0044] In light of this disclosure, it should be recognized that the types of biological materials to be sterilized are extremely diverse and include tissue samples for transplantation or investigation (e.g., corneal tissue, skin, bone, bone marrow, etc.), cellular and decellularized (synthetic and natural) matrices, and even organs for transplantation. Most typically, such tissue samples are mammalian tissues, most typically human tissues (e.g., for autografts or xenografts). Alternatively, the tissues to be sterilized as intended herein also include allografts and even xenografts, and it should be recognized that suitable donor tissues for xenografts include bovine tissue, porcine tissue, lizard tissue, fish tissue, etc., and virtually any tissue suitable for xenografting from one species to another. Furthermore, it should be noted that the sterilization solutions presented herein can also be used to decontaminate biological materials (cells, tissues, cell-containing or cell-free bodily fluids, aspirates, exudates, etc.) before disposal.

[0041]

[0045] Another advantage of the intended use of the sterile solutions is that cells and tissues sterilized by such solutions are substantially unchanged in terms of their biochemical and / or mechanical properties. This is particularly advantageous when the cells or tissues are donor cells or donor tissues to be implanted or transplanted. As used herein, the term “substantially unchanged” means that the change in biochemical and / or mechanical properties is less than 10%. For example, the tensile strength and / or modulus of tendon material does not change by more than 10% in absolute value before and after sterilization. Similarly, the optical transparency of corneal tissue does not change by more than 10% in absolute value before and after sterilization. Similarly, the elasticity of skin does not change by more than 10% in absolute value before and after sterilization. In yet another intended embodiment, the sterile solutions presented herein can also be used as wound cleansing or rinsing solutions for body surfaces (or other media) suspected of microbial contamination. To reiterate, the generation of sterilization solutions at the care / use site is particularly advantageous, especially in field or emergency medical applications. [Examples]

[0042]

[0046] The following examples provide specific guidance for the preparation and use of the sterilization solutions presented herein, but those skilled in the art should recognize that the examples can be modified using the above teachings to obtain further sterilization solutions suitable for use with biological materials.

[0043]

[0047] Exemplary sterilization test

[0044]

[0048] In one embodiment, a dried reagent powder was added to a tissue bath solution to generate in situ a level of NO sufficient to sterilize human tendon tissue. In this embodiment, the inventors tested solutions containing 50–100 mM S-nitrosoglutathione or nitrosocysteine, formed in situ by mixing glutathione (or cysteine) and sodium nitrite in 0.9% physiological saline, phosphate-buffered saline (PBS), and Allowash (commercially available from LifeNet Health, Virginia Beach, VA23453). These solutions were able to sterilize human tendon tissue directly inoculated with E. coli, as will be described in more detail below. After sterilizing the tissue, the sterilization solution was treated with a weak base to decompose residual NO donors, and then neutralized with a weak acid to obtain a salt solution, which was disposed of without requiring chemical disposal requirements.

[0045]

[0049] Human tendon fragments (approximately 2 cm) were immersed in sterile PBS for 20 hours to remove residual antibiotics. Then, 7.4 × 10⁶ of the tendon fragments were applied. 6 100 ml of CFU E. coli was injected and placed in 2 ml of trypsin soy broth (TSB). 2 ml of sterile water was added to the control tendon, and 2 ml of sterile solution (0.9% saline containing 50-100 mM S-nitrosoglutathione or nitrosocysteine, phosphate-buffered saline (PBS), or Allowash) was added to the treatment tendon, and the mixture was gently shaken in an orbital shaker. The tendons were incubated, and samples of the bath solution were taken at 0.5, 1, 2, 3, and 4 hours. A fixed amount of these samples was plated onto Mueller-Hinton agar plates and incubated at 35°C for 48 hours. In particular, as can be seen from Figure 1, no bacteria remained in the tendon solution treated with "sterile solution" after incubation for only 2 hours, which corresponds to a log6 reduction in bacterial load.

[0046]

[0050] After 24 hours, control tendons were placed in 2 mL of fresh TSB and treated with 2 mL of fresh sterile solution. The E. coli load was 10. 8The cyanogenic fungicide (CFU) was estimated. The tendons were incubated with gentle shaking, and samples were taken at 0, 2, 4, 6, and 22 hours. A fixed amount of these samples was plated onto Müller-Hinton agar plates and incubated at 35°C for 48 hours. In particular, as can be seen in Figure 2, all bacteria in the tendons were removed after 6 hours, which corresponds to a log8 reduction in the bacterial load.

[0047]

[0051] Exceptional Mechanical Properties Test

[0048]

[0052] In another embodiment, a tissue sterilization solution was prepared by dissolving 1 g of reduced glutathione and 0.25 g of sodium nitrite in 250 mL of DI water (equivalent to 4 mg / mL of reduced glutathione and 1 mg / mL of sodium nitrite). The solution was rotated / gently agitated until the solids were completely dissolved and used within 2 hours of preparation. The solution thus prepared changed from its initial pink color to red within approximately 5 minutes.

[0049]

[0053] Human tendons were obtained from cerebrospinal fluid (CTS). The tendons were cut along the longitudinal midline. One half was subjected to a tissue sterilization solution, while the other half was immersed in sterile saline solution and used as a control.

[0050]

[0054] Human tendons were transferred to a 50 mL centrifuge tube. To completely submerge the tissue, 30 mL of tissue sterilization solution was added to the centrifuge tube. The tube was then capped and tightly closed. Similarly, control tissue was immersed in 30 mL of sterile saline. Both tubes were placed in a 35°C incubator and incubated for 24 hours.

[0051]

[0055] After processing, all tissues were washed with sterile PBS (phosphate-buffered saline). The tissues were cut into approximately 3-inch long specimens for tensile testing. The dimensions of each specimen are summarized in Table 1, where L indicates the long axis of the tissue cross-section (two positions, L1 and L2, were selected) and W indicates the short axis of the tissue cross-section (two positions, W1 and W2, were selected). Samples 1-4 were treated with a tissue sterilization solution, while controls Ctrl1-4 were treated with sterile saline solution.

[0052] [Table 1]

[0053]

[0056] A standard Construction Materials Testing (CMT) apparatus was used for the mechanical tests. The parameters used for the tensile tests were as follows: sample length (l0) = 20 mm; number of cycles = 30; frequency = 1.5 Hz; length at the start of the cycle = 2 mm; length at the end of the cycle = 4 mm. The following parameters were recorded: time (t, seconds), displacement (Δl, mm), and force (F, N). Stress was calculated as force / area, and strain was calculated as the difference between the distance between the two sample clips at time zero and the distance between the two sample clips at the end / distance between the two sample clips at time zero. The secant coefficient was estimated by calculating the slope of the straight line drawn based on the stress-strain diagram (from strain 14% to 19%). Figure 3 is a photograph of the experimental apparatus.

[0054]

[0057] Figure 4 is a graph showing a comparison of exemplary stress-strain curves, and Table 2 shows the maximum stress and moduli of human tendon samples.

[0055] [Table 2]

[0056]

[0058] A t-test was used to determine whether there was a significant difference between the group treated with the tissue sterilization solution and the control group. The p-values ​​for the secant coefficient and maximum stress were 0.248049 and 0.149467, respectively, indicating no statistically significant difference (p < 0.05). Stress changes over time were measured, and Figure 5 shows an exemplary comparative graph illustrating the stress changes over time during 30 cycles of the tensile test.

[0057]

[0059] In particular, as can be seen from the data above, the tissue sterilization solutions intended herein effectively sterilized human tissue without altering its mechanical properties in a statistically significant manner. Indeed, it should be recognized that the tissue sterilization solutions intended herein do not require high temperatures, high pressures, strong acids, strong bases, or corrosive components, which are typically necessary and under conditions that guarantee sterilization and would significantly alter the mechanical properties of human tissue.

[0058]

[0060] In some embodiments, numbers representing properties such as quantities and concentrations of components, reaction conditions, etc., used to describe and claim certain embodiments of the present invention should be understood to be modified in some cases with the term “about.” As used herein, the terms “about” and “approximately” mean, when referring to a specific measurable value (parameter, quantity, duration, etc.), that specific value and its variation from that specific value, for example, a variation of + / - 10%, or + / - 5%, or + / - 1%, or + / - 0.1%, from that specific value, but only if such variation is appropriate for the implementation of the disclosed embodiments. Thus, the values ​​themselves that the modifiers “about” or “approximately” refer to are also specifically disclosed. The descriptions of value ranges herein are merely intended to serve as a simple way to refer individually to each individual value within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually stated herein. Unless otherwise noted or indicated by the context, all percentages shown are weight percentages.

[0059]

[0061] All methods described herein may be performed in any suitable order, unless otherwise indicated herein or unless it is clearly inconsistent with the context. Any and all examples or illustrative expressions (e.g., "etc.") provided herein in reference to any embodiment are intended solely to further illustrate the invention and, unless otherwise claimed, do not limit the scope of the invention. Terms herein should not be construed as indicating any unclaimed element essential to the practice of the invention.

[0060]

[0062] As used herein in this description and in the claims, “a,” “an,” and “the” include plural references unless explicitly indicated otherwise by the context. Similarly, as used herein, “in” includes “in” and “on” unless explicitly indicated otherwise by the context. Furthermore, as used herein, unless explicitly indicated otherwise by the context, the term “coupled to” is intended to include both direct coupling (two coupled elements touching each other) and indirect coupling (at least one additional element positioned between two elements). Therefore, the terms “coupled to” and “coupled with” are used as synonyms.

[0061]

[0063] It will be apparent to those skilled in the art that, without departing from the inventive concept herein, many more modifications are possible beyond those already described. Therefore, the subject matter of the present invention is not limited beyond the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible way that is appropriate to the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the element, component, or step referred to may exist with, be used with, or be combined with other elements, components, or steps not specifically mentioned. Where the specification or claims refer to at least one of any selected from the group consisting of A, B, C…, and N, the wording should be interpreted as requiring only one element from the group, rather than A and N or B and N, etc.

Claims

1. A method for sterilizing biological tissue, The number of bacteria in and / or on the aforementioned tissue is at least 10 ４ The step is to leave the tissue in a sterilization solution for a sufficient time to reduce it to one-tenth, The aforementioned tissue is donor tissue for transplantation. The sterilization solution contains a nitric oxide precursor material that decomposes in place and releases a sufficient amount of nitric oxide to reduce the number of bacteria. A method comprising the step of generating the nitric oxide precursor material in situ by reacting a first reagent and a second reagent in the solution.

2. The method according to claim 1, wherein the tissue is human donor tissue.

3. The method according to claim 2, wherein the tissue is a tendon, cornea, bone, or skin.

4. The method according to claim 1, wherein the sterilization solution is an isotonic aqueous solution.

5. The method according to claim 4, wherein the aqueous solution further comprises at least one of an osmotic pressure molar concentration adjusting agent, nutrients, and a cleaning agent.

6. The method according to claim 1, wherein the time is 60 to 2800 minutes.

7. The method according to claim 1, wherein the tissue is sterilized in the solution at a temperature of 4°C to 37°C, and / or the tissue is sterilized in the solution at a pressure of 900 to 1,100 mbar.

8. The number of bacteria is at least 10 ６ The method according to claim 1, wherein the amount is reduced to 1 / 2.

9. The number of bacteria is at least 10 ８ The method according to claim 1, wherein the amount is reduced to 1 / 2.

10. The method according to claim 1, wherein the tissue is a tendon, the change in the tissue parameters of the tendon after sterilization is 10% or less, and the tissue parameters are selected from the group consisting of fracture load, ultimate tensile strength, and Young's modulus.

11. The method according to claim 1, wherein the nitric oxide precursor material comprises a nitrosothiol.

12. The method according to claim 11, wherein the nitrosothiol is S-nitrosoglutathione or nitrosocysteine.

13. The method according to claim 1, wherein the nitric oxide precursor material contains a nitrosothiol, and the first reagent and the second reagent are a thiol-containing compound and a nitrosated compound, respectively.

14. The method according to claim 13, wherein the thiol-containing compound and the nitrosated compound are provided in the form of a dry compound that, when added to a solution, forms the sterilization solution.

15. A method for sterilizing tendon tissue while maintaining the mechanical properties of the tendon tissue, The number of bacteria in and / or on the aforementioned tissue is at least 10 ４ The step is to leave the tendon tissue in a sterile solution for a sufficient time to reduce it to one-tenth, The solution contains a nitric oxide precursor material that decomposes in place and releases a sufficient amount of nitric oxide to reduce the number of bacteria, A method comprising the step of generating the nitric oxide precursor material in situ by reacting a first reagent and a second reagent in the solution.

16. The method according to claim 15, wherein the sterilization solution is an aqueous solution, the nitric oxide precursor material contains a nitrosothiol, and the first reagent and the second reagent are a thiol-containing compound and a nitrosated compound, respectively.

17. The method according to claim 16, wherein the thiol-containing compound and the nitrosated compound are provided in the form of a dry compound that, when added to a solution, forms the sterilization solution.

18. The method according to claim 17, wherein the dried compound is a tablet or a powder.

19. The method according to claim 15, wherein the tissue is sterilized in the solution at a temperature of 4°C to 37°C, and / or the tissue is sterilized in the solution at a pressure of 900 to 1,100 mbar.

20. The number of bacteria is at least 10 ６ The method according to claim 15, which reduces to 1 / 2.

21. The method according to claim 15, wherein the mechanical properties are selected from the group consisting of breaking load, ultimate tensile strength, and / or Young's modulus.

22. Biological tissue immersed in a sterile solution, wherein the sterile solution contains a nitric oxide precursor material that decomposes in the solution to release a sufficient amount of nitric oxide to reduce the number of bacteria in and / or on the tissue; A first reagent and a second reagent, wherein a reaction between the first reagent and the second reagent in situ forms a nitric oxide precursor material. It is an intermediate sterilization composition containing, The nitric oxide precursor material reduces the number of bacteria in and / or on the tissue by at least 10 after the tissue has been exposed to nitric oxide for 6 hours or less. ４ An intermediate sterilization composition having a composition that releases an amount of nitric oxide that reduces the amount to one-tenth.

23. The sterilization composition according to claim 22, wherein the nitric oxide precursor material is nitrosothiol.

24. The sterilization composition according to claim 22, wherein the biological tissue is sterilized in the solution at a temperature of 4°C to 37°C, and / or the tissue is sterilized in the solution at a pressure of 900 to 1,100 mbar.

25. The sterilization composition according to claim 22, wherein the change in the mechanical properties of the biological tissue is 10% or less before and after sterilization.

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