Systems and methods for improved sterile processing of biological material

Abstract

Products, processes, compositions, kits, and methods are provided for cartilage-derived implants. The implants can exhibit resistance to enzyme (e.g., collagenase, protease, etc.) digestion compared to the source tissue from which they were derived while still having one or more mechanical properties comparable to the source tissue from which they were derived. The implants can be processed in a manner that prevents formation or reduces the size of pores in the implants. The implants can also have a plurality of molecular bridges between molecules of the cartilaginous material. The molecular bridges can connect one or more collagen fibrils and / or / with one or more glycosaminoglycans. The implants can also be treated with cationic detergent, packaged and sterilized with or without additional components, and surgically implanted into subjects.

Description

SYSTEMS AND METHODS FOR IMPROVED STERILE PROCESSING OF BIOLOGICAL MATERIALBACKGROUND1. Technical Field

[0001] The present invention relates to cartilage-derived materials, methods of sterilization, and methods of production. More specifically, the present invention relates to a method of creating a sterile, cartilage-derived material that has a uniform structure.2. Relevant Technology

[0002] Cartilage is a solid connective tissue that sen es as a shock absorbing surface in our joints; provides structure to the rib cage, trachea, ears and nose; and provides cushioning between the vertebrae of our spine. When cartilage is damaged by injury or disease, the human body has little ability to regenerate the damaged cartilage and there are no ideal biomaterial cartilage replacement materials available clinically for repair. The lack of ideal cartilage repair biomaterials is why spinal fusion is the ultimate treatment for intervertebral disc degeneration, why metal implants are the standard of repair for severe cartilage degeneration in the hip and knees, and why cartilage graft harvesting is the most common source of cartilage in ear and nose repair. Therefore, there is a clinical need for improved biological cartilage repair materials in all anatomical locations.

[0003] Animal cartilage (both human and non-human) can be a potential source of cartilage material from which cartilage repair materials can be derived. The ability to use a cartilage material for medical purposes requires a method of sterilization. Irradiation of a cartilage material can be a useful method of sterilization.BRIEF SUMMARY

[0004] The present invention relates to cartilage-derived tissue that has been processed to be biocompatible for human implantation. For instance, an embodiment includes a cartilage- derived implant that is essentially void of living cells (from the source organism), resistant to cellular infiltration, optionally resistant to enzymatic resorption, and / or mechanically comparable in feel and / or strength compared to the source cartilage material. In at least one embodiment, the source cartilage can be rapidly frozen with methods described below to prevent the formation or reduce the size of pores formed in the cartilage derived tissue during the freezing process and / or present after thawing the frozen cartilage derived tissue.

[0005] At least one embodiment involves creating a crosslinked cartilage material from a source cartilage or obtaining and already crosslinked source cartilage, rapidly freezing thecrosslinked cartilage to prevent ice cry stal formation and irradiating the crosslinked cartilage while maintaining a frozen state throughout the irradiation.

[0006] At least one embodiment includes a cartilage-derived material. The cartilage-derived material can comprise intact extra-cellular matrix of a source cartilage. In one or more embodiments, the cartilage-derived material, or the intact extra-cellular matrix thereof, can have or exhibit an increased resistance to enzymatic degradation from an extracellular matrix degrading enzyme compared to the source cartilage.

[0007] In one or more embodiments, the cartilage-derived material, or the intact extracellular matrix thereof, can have or exhibit sterility gained by irradiating the cartilage-derived material while in a frozen state.

[0008] In one or more embodiments, the cartilage-derived material, or the intact extracellular matrix thereof, can have or exhibit an absence of visible pores or pores visible with the naked eye (e.g., pores that would have been present if, prior to irradiation, the cartilage material was frozen at a slower rate) or pores of a reduced size as compared to the size of the pores if, prior to irradiation, the cartilage material was frozen at a slower rate. In other words, embodiments of the present disclosure include cartilage material that (i) is devoid of visible pores or pores visible to the naked eye, (ii) does not exhibit visible pores or pores visible to the naked eye that are present in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed, and / or (iii) has or exhibits visible pores or pores visible to the naked eye that are of a reduced size as compared to the size of pores in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed.

[0009] In some embodiments, the cartilage-derived material, relative to the source cartilage, can comprise a reduced amount of one or more components selected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins; and galactose-alpha-1 ,3-galactose.

[0010] The present invention also relates to methods of processing animal-derived cartilage into a cartilage-derived implant possessing one or more of the characteristics described herein. In some embodiments, the method comprises treating the cartilage with a cationic detergent solution. In some embodiments, the method comprises treating the cartilage with a stabilizing agent that forms molecular bridges within the cartilage-derived implant. Some embodiments include a (terminal) sterilization step (e.g., gamma and / or electron beam irradiation).

[0011] At least one embodiment includes a method for preparing a cartilage-derived material. In some embodiments, the method comprises a step of obtaining source cartilage comprising intact extra-cellular matrix. In some embodiments, the source cartilage, or intact extra-cellular matrix thereof, has been treated to form treated cartilage. In some embodiment, the intact extra-cellular matrix or treated cartilage has an increased resistance to enzymatic degradation by an extra-cellular matrix degrading enzyme as compared to the source cartilage.

[0012] In some embodiments, the method comprises a step of freezing the treated cartilage to form frozen, treated cartilage.

[0013] In some embodiments, the method comprises a step of irradiating the frozen, treated cartilage while in a frozen state to form sterilized, frozen, treated cartilage.

[0014] In some embodiments, the source cartilage is derived from an animal or lab-grown source. In some embodiments, the animal source cartilage comprises an explant from a pig, horse, cow, canine, human, or non-human animal and is selected from the group consisting of septal cartilage, auricular cartilage, costal cartilage, and articular cartilage.

[0015] In some embodiments, the step of freezing the treated cartilage involves submersion into liquid nitrogen, submersion into a mix of alcohol and dry ice, exposure to liquid nitrogen sprays, direct contact with dry ice, or placement in -80C freezer wherein the cartilage material is placed directly on a first precooled metal pan and a second precooled metal pan is placed on top of the cartilage material. In some embodiments, the step of freezing is performed in less than 70 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In some embodiments, the step of freezing the treated cartilage reduces the temperature of the treated cartilage from greater than 0 degrees Celsius to less than or equal to 0 degrees Celsius in less than 70 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In some embodiments, the step of freezing the treated cartilage reduces the temperature of the treated cartilage from greater than or equal to 4 degrees Celsius to less than or equal to 0 degrees Celsius in less than 70 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In some embodiments, the step of freezing the treated cartilage reduces the temperature of the treated cartilage to less than or equal to -72 degrees Celsius.Some embodiments include a further step of thawing the sterilized, frozen, treated cartilage after irradiation to obtain an irradiated, enzyme-resistant cartilage material. In some embodiments, after the thawing step, (i) the irradiated, enzyme-resistant cartilage material is devoid of visible pores or pores visible to the naked eye, (ii) the irradiated, enzyme-resistantcartilage material does not exhibit visible pores or pores visible to the naked eye that are present in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed, and / or (iii) visible pores or pores visible to the naked eye in the irradiated, enzyme-resistant cartilage material are of a reduced size as compared to the size of pores in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed. In some embodiments, visible pores are not present in the material that would be present if the same material were frozen at a slower rate. In some embodiments, visible pores in the material are of a reduced size as compared to the size of pores in material frozen at a slower rate.

[0016] In some embodiments, the treated cartilage has been treated with a cationic detergent. In some embodiments, the cationic detergent comprises one or more components selected from the group consisting of a quaternary ammonium compound, myristyltrimethylammonium bromide, cetyltrimethylammonium bromide, tridodecylmethylammonium chloride, dodecyltrimethylammonium bromide, hexadecylpyridinium chloride, cetylpyridinium chloride, benzyldodecyldimethylammonium bromide, and combinations thereof.

[0017] In some embodiments, the treated cartilage has been treated with at least one multifunctional reactive molecule configured to form one or more molecular bridges between molecules of the source cartilage, the one or more molecular bridges selected from the group consisting of: a hydrocarbon diol-containing bridge having between 3 and 12 carbon atoms; a sulfone-containing bridge; a molecular bridge between amine groups of one or more collagen fibrils; a molecular bridge between hydroxyl groups of one or more glycosaminoglycan molecules; and a molecular bridge between a hydroxyl group of a glycosaminoglycan molecule and an amine group of a collagen fibril. In some embodiments, the at least one multifunctional reactive molecule is selected from the group consisting of: a molecule comprising more than one and less than 5 reactive epoxide groups; divinyl sulfone; I. 4 butanediol diglycidyl ether; 1, 2, 7, 8 diepoxyoctane; and combinations thereof. In some embodiments, the at least one multifunctional reactive molecule is provided in solution at a concentration of between 0. 1 mM and 100 mM. In some embodiments, the treated cartilage is resistant to enzymatic digestion compared to the source cartilage.

[0018] In some embodiments, the step of irradiating the frozen, treated cartilage while in a frozen state comprises exposing the cartilage-derived implant sealed in a radiation resistant package to at least one dose of electron beam radiation between 1 and 20 kGy and at a temperature of between -20°C and -80°C.

[0019] At least one embodiment includes a cartilage derived material prepared by the method, above.

[0020] At least one embodiment includes a method for preparing a cartilage-derived material, comprising: obtaining source cartilage from an animal or lab grown source, the source cartilage comprising intact extra-cellular matrix; optionally treating the source cartilage so as to remove an amount of one or more components selected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins: and galactose-alpha-l,3-galactose; treating the source cartilage to be more resistant to enzymatic degradation from an extracellular matrix degrading enzyme compared to the source cartilage without treatment; freezing the treated cartilage at a rate that results in: (i) an absence of visible pores, or pores visible with the naked eye, in the material that would have been present if the same material were frozen at a slower rate; and / or (ii) pores of a reduced size in the cartilage material as compared to the size of the pores had the material been frozen at a slower rate; optionally irradiating the cartilage-derived material in the frozen state; and optionally thawing the cartilage material.

[0021] In some embodiments, the method further includes one or more steps selected from the group of steps consisting of: rinsing the source cartilage to remove cellular remnants; treating the source cartilage with a solution comprising a cationic detergent; rinsing the source cartilage to remove at least some cationic detergent; rinsing the cartilage-derived implant with a solution comprising a primary amine-containing solute; sealing the cartilage-derived implant in a package; sealing the cartilage-derived implant in a radiation resistant package; sterilizing the cartilage-derived implant sealed in a radiation resistant package by irradiation; seeding the cartilage-derived implant with living human cells; sculpting the cartilage-derived implant; and implanting the cartilage-derived implant into a subject. In at least one embodiment, the subject is mammalian, human, or a non-human mammal.

[0022] The present invention also relates to methods of use for a cartilage-derived implant. In some embodiments, the cartilage-derived implant is used for repairing or reconstructing cartilaginous structures. In some embodiments the method comprises obtaining the cartilage- derived implant described herein, sculpting the cartilage-derived implant into a specific shape,and / or implanting the cartilage-derived implant into an anatomical location (e.g., a nose or ear requiring repair with a cartilage-derived implant).BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A illustrates an exemplary reduction in residual DNA following cationic detergent treatment according to an embodiment of the present invention.

[0024] Figure IB illustrates an exemplary retention in compressive yield stress following cationic detergent treatment according to an embodiment of the present invention.

[0025] Figure 2A illustrates an exemplary crosslinking reaction according to an embodiment of the present invention.

[0026] Figure 2B illustrates another exemplary crosslinking reaction according to an embodiment of the present invention.

[0027] Figure 3A illustrates an exemplary increase in collagenase stability following crosslinking treatment according to an embodiment of the present invention.

[0028] Figure 3B illustrates an exemplary retention in compressive yield stress following crosslinking treatment according to an embodiment of the present invention.

[0029] Figure 4 illustrates an exemplary method of preparing a cartilage-derived implant according to an embodiment of the present invention.

[0030] Figure 5 illustrates an exemplary' series of reductions in residual DNA following cationic detergent treatment according to an embodiment of the present invention.

[0031] Figure 6 illustrates an exemplary series of percent reduction in DNA content following cationic detergent treatment according to an embodiment of the present invention.

[0032] Figure 7 illustrates exemplary' residual galactose-alpha-l,3-galactose following cationic and zwitterionic detergent treatment according to an embodiment of the present invention.

[0033] Figure 8 illustrates an exemplary series of retention in compressive yield stress following cationic detergent treatment and crosslinking treatment according to an embodiment of the present invention.

[0034] Figure 9 illustrates an exemplary series of collagenase resistance following crosslinking treatment according to an embodiment of the present invention.

[0035] Figure 10 illustrates the unexpected problem of visible pore formation that occurs during freezing.

[0036] Figure 11 A depicts aspects of an exemplary method of rapidly freezing cartilage and Figure 1 IB depicts aspects of an exemplary method of slow freezing cartilage.

[0037] Figures 12A-12D illustrate an exemplary' reduction in pore formation when rapid freezing a source cartilage compared to various methods of slower freezing of a source cartilage.

[0038] Figures 13 A and 13B illustrate an exemplary reduction in pore formation when rapid freezing a source cartilage after a cartilage sample has been irradiated and thawed, compared to slow freezing a source cartilage.DETAILED DESCRIPTION

[0039] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to parameters of the particularly exemplified products, processes, compositions, kits, and / or methods, which may, of course, vary. It is also to be understood that the terminology used herein is only for the purpose of describing particular embodiments of the present invention and is not intended to limit the scope of the invention in any manner.

[0040] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0041] Additionally, the terms “including,” “having,” “involving,” “containing,” “characterized by,” and variants thereof (e.g, “includes,” “has,” and “involves,” “contains,” etc.) as used herein, including the claims, shall be inclusive and / or open ended, shall have the same meaning as the word “comprising” and variants thereof (e.g., “comprise” and “comprises”), and does not exclude additional, unrecited elements or method steps, illustratively.

[0042] It will be noted that, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a “support member” includes one, two, or more support members.

[0043] Various aspects of the present invention may be illustrated with reference to one or more exemplary embodiments. As used herein, the term “exemplary” means “serving as an example, instance, or illustration.” and should not necessarily be construed as preferred or advantageous over other embodiments disclosed herein.

[0044] It will also be appreciated that where a range of values (e.g., less than, greater than, at least, or up to a certain value, or between two recited values) is disclosed or recited, any specific value or range of values falling within the disclosed range of values is likewise disclosed and contemplated herein. Thus, disclosure of a pH between 5 and 9 includes,illustratively a specific disclosure of: (i) a pH of 5.2, 6.5, 8, or any other value between 5 and 9; and / or (ii) a pH between 5 and 8, a pH between 6 and 9, a pH between 7 and 8, and / or any other smaller range of values between 5 and 9.

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. While a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present disclosure, only preferred materials and methods are described herein.

[0046] The present relates to processes and methods of producing a sterile, crosslinked cartilage-derived material that has a uniform structure. The invention involves creating a crosslinked cartilage material from a source cartilage or obtaining and already crosslinked source cartilage, rapidly freezing the crosslinked cartilage to prevent ice crystal formation, and irradiating the crosslinked cartilage while maintaining a frozen state throughout the irradiation.

[0047] Irradiation of a frozen cartilage material (at frozen temperatures) can be a useful method of sterilization. However, some cartilage materials can change form or be physically altered when frozen (or by freezing). For example, in the case of a crosslinked cartilage, freezing the material by standard methods, such as placing the material in a -20 C or -80 C freezer, can result in ice cry stal formation. Because crosslinked cartilage has altered physical bonds compared to the source cartilage, sometimes these ice crystals can grow to create (large and / or visible) pockets or pores in the cartilage material. Normally, when the crosslinked cartilage material that has pores is subsequently thawed, the ice crystals melt and the crosslinked cartilage material can expand to its original state without visible pores. However, when these pores are present in a frozen cartilage material and the cartilage material is subsequently irradiated (while the cartilage material is frozen), then the irradiation can cause the material to become fixed (e.g., crosslinked) in place, thus making the pores fixed in place even after thawing. This is further supported by KR20200018793A, which describes how electron beam radiation can be used to both sterilize a hyaluronic acid gel and crosslink the gel at the same time. In this case, the crosslinking was described as being beneficial because the radiation mediated crosslinking can remove any unreacted end groups of residual crosslinker in solution, reducing toxicity7and improving physical properties at the same time.

[0048] When a cross-linked cartilage sample is frozen, large ice crystals can form that present as large pores in the cartilage material. The large pores caused by ice formation typically disappear upon thawing of the material because the ice crystals that create the pores melt and the material will expand back to occupy the pore space. However, when the cartilagematerial containing pores is irradiated while frozen, both radiation-mediated physical crosslinking and any residual crosslinker or unreacted crosslinker pendant groups are activated to create new crosslinks within the cartilage material. The radiation causes the material to be further cross-linked in place and the large pores formed by the ice crystals are locked in place. Upon thawing of the irradiated cartilage material, the cartilage implant presents itself with large visible pores. The pores do not disappear upon thaw ing and the cartilage material does not reexpand to occupy the space created by the ice crystals. These large pores render the material non-uniform and not ideal for some purposes.

[0049] While it could be possible to minimize this problem by significantly lengthening the initial cartilage material cross-linking reaction time and the cross-linker removal by dilution time thereby reducing the quantity of any pendant ends or residual crosslinker, that solution would introduce new problems associated with adding days to the processing, such as potential damage to sensitive cartilage extracellular matrix molecules and increased cost of production. In addition, this would not prevent radiation-mediated crosslinking.

[0050] The proposed embodiments described hereinbelow prevent the presence of pores in a cartilage-derived material following irradiation and remove unreacted crosslinker ends without having to perform extended rinses and reaction times. The invention involves freezing the cartilage material at a fast enough rate to prevent the formation of ice cry stals prior to irradiating the cartilage material. The invention of rapid freezing the crosslinked cartilage material prior to irradiation prevents the presence of pores following irradiation, allows for the irradiation to eliminate unreacted ends without lengthy reaction and dilution times, and provides an additional benefit of further stabilizing the cartilage in its desired state without large visible pores.

[0051] The invention of rapid freezing a crosslinked cartilage material prior to irradiation (or any other process that can crosslink a frozen cartilage material) prevents the formation of pores following irradiation, allows for the irradiation to eliminate unreacted ends without lengthy reaction and dilution times, and provides an additional benefit of further stabilizing the cartilage in its desired 3D state without large visible pores.

[0052] In some embodiments, cartilage-derived materials can be prepared as described in patents US10016528B2 and USI0814035B2, the entirety of each of which are incorporated herein by specific reference. Cartilage-derived materials prepared in this manner may have antigenic remnants from the source cartilage removed and the cartilage materials may be crosslinked using multifunctional crosslinkers. In some embodiments, there may be residual crosslinker or residual unreacted end groups present in the crosslinked cartilage material priorto irradiation. In some embodiments the cartilage is treated to remove antigenic remnants prior to being crosslinked and prior to being frozen.

[0053] The present invention relates to a cartilage-derived implant that has been processed to be biocompatible for or upon human implantation. For instance, the cartilage-derived implant can be sterile within a defined sterility assurance level. The cartilage-derived implant can also have or exhibit a reduced level (or be essentially void or free) of residual source organism (living) cells and / or cellular remnants compared to the source cartilage. Accordingly, the chance of a non-allergic foreign body rejection of the cartilage-derived implant upon implantation (e.g., into a human) can be reduced or eliminated.

[0054] The cartilage-derived implant can also be resistant to or against cellular infiltration and / or enzymatic digestion (e.g.. degradation and / or resorption). The cartilage-derived implant can also be biomechanically and / or biochemically comparable to the source cartilage material. For instance, the cartilage-derived implant can have or exhibit increased stability compared to the source cartilage while retaining mechanical properties substantially similar to the source cartilage such that the cartilage-derived implant is comparable in feel and / or mechanical property to natural cartilage when it is implanted in a body.

[0055] The cartilage-derived implant can also be processed to retain a uniform structure. For instance, the cartilage-derived implant can be processed in a manner to prevent formation of large pores which may negatively affect the biocompatibility for or upon human implementation. The cartilage-derived implant can also have or exhibit a reduced level (or be essentially void or free) pores. Accordingly, one will appreciate that reference to '‘processed cartilage” and similar terms can refer to a cartilage-derived implant that has been processed to effectuate any one or more of the above-identified or other characteristic or properties. In at least one embodiment, the source cartilage can be derived from the septal, auricular, costal, and / or articular cartilage of a pig, horse, cow, canine, human, or other animal.

[0056] One will appreciate that reference to an implant is exemplary, and various configurations, designs, intended uses, etc. are known in the art and contemplated herein. For purposes of illustration only, this disclosure refers to implants designed for human implantation. One will appreciate, however, that non-human implantation and nonimplantation uses for the cartilage-derived tissue disclosed herein are also within the scope of this disclosure.

[0057] As will be discussed in further detail below, processing cartilage (e.g., to effectuate any one or more of the above-identified or other characteristic or properties) can be achieved by treatment with a cationic detergent (e.g., to remove source cells and / or cellular remnants).Accordingly, in one or more embodiments, the cartilage-derived implant (or composition thereof) can comprise or be associated with at least a residual or trace amount of a cationic detergent. In at least one embodiment, cationic detergent treatment can remove an amount of living cells and / or cellular remnants from the source cartilage.

[0058] In some embodiments, the processed, cartilage-derived implant may include an amount of: (i) living cells; (ii) living cells of the donor or source organism; and / or (iii) cellular remnants, the amount being sufficiently small such that the chance of a non-allergic foreign body rejection upon implantation (e.g., into a human) is reduced compared to the source cartilage. In some embodiments, the cartilage-derived implant may include an amount of: (i) living cells; (ii) living cells of the donor or source organism; and / or (iii) cellular remnants, the amount being within (agency) standards (e.g.. FDA) for the cartilage-derived implant (e.g., to be used as a medical device or implant).

[0059] For instance, in some embodiments, the cartilage-derived implant is essentially void of living cells (e.g., of the donor or source organism) and / or cellular remnants. As used herein, “essentially void” and similar terms can refer to undetectable amounts (by one or more, or all, current detection methods). In addition, “essentially void” and similar terms can refer to less than 0.01%, less than 0.1%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% compared to an original amount or control amount of the component present in the source cartilage or explant.

[0060] In some embodiments, the cartilage-derived implant comprises a reduced amount of living cells and / or cellular remnants (e.g., compared to the source cartilage). As used herein, “reduced amount” and similar terms can refer to any statistically significant decrease in an amount compared to a standard (e.g., the source cartilage from which a cartilage-derived implant was derived). For instance, in some embodiments, the cartilage-derived implant can comprise or retain less than 0.0001%, less than 0.001%, less than 0.01%, less than 0.1%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, or less than 35% of living cells and / or living cells of the donor or source organism. Similarly, in certain embodiments, the cartilage-derived implant has been processed in order to remove at least some of the living cells and / or cellular remnants of or from the source cartilage.

[0061] As used herein, “cellular remnants” and similar terms can refer to DNA, RNA, soluble proteins, immunogenic proteins, immunogenic effectors, major histocompatibilitycomplex (MHC) proteins. galactose-alpha- l .3-galactose. blood, and / or cellular vestiges or debris of the donor or source organism (e.g., not including structural components of cartilage and / or the extra-cellular matrix (ECM) of the donor or source organism). In at least one embodiment, a cartilage-derived implant can exhibit up to, at least, greater than, or approximately a 400-fold decrease in the level or amount of residual, source organism DNA (w / w) compared to the source cartilage. For instance, as illustrated in Fig. 1A, native, source cartilage can comprise approximately 260 ng DNA per mg wet weight cartilage. Processed cartilage (i.e., a cartilage-derived implant), on the other hand, can comprise approximately 13 ng DNA per mg wet weight cartilage. Thus, a cartilage-derived implant can exhibit up to, at least, greater than, or approximately a 95% reduction in the level or amount of source organism DNA (w / w) compared to the native, unprocessed, source tissue (p=1.012).

[0062] One will appreciate, however, that other levels of DNA reduction (or amounts of residual DNA) are contemplated herein. For instance, a processed, cartilage-derived implant can exhibit up to, at least, greater than, or approximately a 2-fold, 3-fold, 4-fold, 5-fold, 10- fold, 20-fold, 50-fold. 100-fold, 250-fold, 500-fold, or 1000-fold decrease in the level or amount of residual, source organism DNA (compared to the source tissue). Likewise, processed tissue can exhibit greater than a 25%, 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% decrease in the amount of source organism DNA (compared to the source tissue), or any value or range therebetween. One will also appreciate that different tissue samples and / or different organisms can exhibit varying amounts of DNA in their native, unprocessed state.

[0063] In at least one embodiment, the amount of residual DNA associated with a cartilage- derived implant can represent the amount of living cells and / or cellular remnant(s) associated with a cartilage-derived implant. For instance, in some embodiments, residual DNA levels can provide an indication of the amount of living cells and / or cellular remnants present in the cartilage-derived implant or other cartilaginous sample. In particular, the amount of residual DNA can represent the amount immunogenic proteins, immunogenic effectors, galactose- alpha-l,3-galactose, and / or major histocompatibility complex (MHC) proteins, which can trigger an immune response in the implant host or patient. Accordingly, in at least one embodiment, the cartilage-derived implant comprises a reduced amount of one or more immunogenic proteins, immunogenic effectors, galactose-alpha-l,3-galactose, and / or major histocompatibility complex (MHC) proteins, compared to the cartilage explant or unprocessed cartilage. For instance, in some embodiments, the cartilage-derived implant has been processed in order to remove some or essentially all galactose-alpha-l,3-galactose from the sourcecartilage explant. Furthermore, as discussed in further detail below, in some embodiments, cationic detergent treatment can have or provide the advantage of more effectively removing galactose-alpha-l,3-galactose and / or MHC proteins from the source cartilage compared to other non-cationic detergent washes.

[0064] In some embodiments, the source cartilage is derived from an animal genetically deficient in expression of galactose-alpha-l,3-galactose. Likewise, in some embodiments, the source cartilage is derived from an animal that expresses reduced levels of galactose-alpha-1,3- galactose. For instance, in some embodiments, the source cartilage is derived from an animal (e.g., a non-human animal) genetically altered to express no or reduced levels of galactose- alpha-l,3-galactose. In some embodiments, the source cartilage is derived from an animal genetically altered to express one or more human proteins. In some embodiments, the cartilage- derived implant comprises a reduced amount and / or is essentially void of both cellular remnants and galactose-alpha-l,3-galactose.

[0065] In at least one embodiment, cationic detergent treatment can remove an amount of living cells and / or cellular remnants from the source cartilage while not significantly altering one or more biochemical and / or biomechanical properties of the cartilage. Accordingly, in some embodiments, the cartilage-derived implant has or exhibits one or more comparable biomechanical properties relative to the source cartilage. For instance, the cartilage-derived implant can have or exhibit comparable biomechanical strength (e.g., as measured by compressive yield stress), compressibility, yield strain at break, elasticity, instantaneous stiffness, tensile strength, tensile strain, coefficient of friction, resilience, shock absorption, feel, Young’s modulus, and / or other biomechanical properties relative to the source cartilage. Likewise, cationic detergent treatment can remove an amount of living cells and / or cellular remnants from the source cartilage while leaving the extra-cellular matrix structure of the cartilage substantially intact.

[0066] In some embodiments, "comparable" (e.g., biomechanical properties) ‘'substantially the same,” and similar terms can refer to and / or be defined as: a measurement value that falls within the range of the natural variation of that measurement value for the source cartilage; a measurement value within or less than 0.1%, 1%. 2%, 3%, 4%. 5%, 10%, 15%, 20%, or 25% of the average or range of the natural variation of that measurement value for the source cartilage; and / or a measurement value within a statistically relevant deviation or error value for the range of the natural variation of that measurement value for the source cartilage. For instance, a value that is comparable or substantially the same as one or more reference values can be within one standard deviation of the average of the reference values.

[0067] As illustrated in Fig. IB, processed, cationic detergent-treated cartilage can retain a comparable level of biomechanical (compressive yield) strength (relative to the native, unprocessed, source cartilage). In the illustrated embodiment, native, unprocessed, source cartilage has a compressive yield stress value of approximately 5.9 MPa (Std. Dev. = 1.6) and processed, cationic detergent-treated cartilage has a compressive yield stress value of approximately 5.7 MPa (Std. Dev. = 2.0). Thus, the compressive yield stress value of the processed, cationic detergent-treated cartilage falls within the range of the natural variation of the compressive yield stress value for the source cartilage or within a statistically relevant deviation or error value for the range of the natural variation of the compressive yield stress value for the source cartilage. Thus, processed cartilage-derived implants can retain one or more biomechanical properties of the source cartilage (e.g., while having a reduced level of source cartilage explant-associated cells and / or cellular remnants).

[0068] In at least one embodiment, the cartilage-derived implant has or exhibits one or more comparable biochemical properties (e.g., relative to the source cartilage). For instance, the cartilage-derived implant can have or exhibit a molecular composition comparable to the source cartilage. In some embodiments, the cartilage-derived implant can retain one or more vital biochemical components of cartilage (e.g., glycosaminoglycans (GAGs) and / or (type II) collagen). One will appreciate that certain embodiments of the present invention can comprise a cartilage-derived implant having one or more biochemical components of cartilage in an altered or modified biochemical or molecular form (e.g., structure) without departing from the scope of this disclosure. For instance, as will be discussed in further detail below, embodiments of the present invention can comprise a cartilage-derived implant having crosslinked GAGs and / or collagen (fibrils).

[0069] In some embodiments, the cartilage-derived implant comprises one or more molecular bridges or crosslinks between the macromolecules that comprise the source cartilage. In at least one embodiment, the macromolecules that comprise cartilage include GAGs and proteins, including collagen. Referring now to Fig. 2A, some embodiments comprise one or more sulfone-containing bridges that are covalently bonded to a GAG by an ether bond and / or covalently bonded to protein by a secondary amine bond. In certain embodiments, one or more sulfone-containing bridges can link: (i) a hydroxyl group of a first GAG to an adjacent hydroxyl group of the same or different GAG; (ii) a hydroxyl group of a GAG to an adjacent amine group of an adjacent protein (e.g., collagen); or (iii) an amine group of a protein to an adjacent amine group of the same or different protein.

[0070] For illustrative purposes only, Fig. 2A illustrates cartilage 5, comprising two collagen fibrils 10 and two GAG molecules 20. Collagen fibril 10a has two free amine groups and collagen fibril 10b has three free amine groups available to form bridges. Similarly, GAG 20a has three free hydroxyl groups and GAG 20b has two free hydroxyl groups available to form bridges. Fig. 2A also illustrates crosslinker 30. Crosslinker 30 can comprise a sulfone (e.g., divinyl sulfone or another reactive compound operable for generating one or more sulfone-containing bridges between molecules of cartilage 5).

[0071] Following reaction 70, crosslinked cartilage 50 comprises: (i) a sulfone-containing bridge 80a between a hydroxyl group of a first GAG and a hydroxyl group of a second GAG;(ii) a sulfone-containing bridge 80b between a hydroxyl group of a GAG and an amine group of a collagen fibril; (iii) a sulfone-containing bridge 80c between an amine group of a first collagen fibril and an amine group of a second collagen fibril; (iv); a sulfone-containing bridge 80d between amine groups of the second collagen fibril; and (v) a sulfone-containing bridge 80e between hydroxyl groups of the second GAG. One will appreciate that in certain embodiments, reaction conditions can promote the formation of one or more specific type(s) of sulfone-containing bridges 80.

[0072] Referring now to Fig. 2B, some embodiments comprise one or more diol -containing bridges that are covalently bonded to a GAG by an ether bond and / or covalently bonded to a protein by a secondary amine bond. In one or more embodiments, diol-containing bridges can link: (i) a hydroxyl group of a GAG to an adjacent hydroxyl group of the same or different GAG; (ii) a hydroxyl group of a GAG to an adjacent amine group of an adjacent protein; or(iii) an amine group of a protein to an adjacent amine group of the same or different protein.

[0073] Some embodiments comprise one or more diol-containing bridges having between 3 and 12 carbon atoms. For instance, Figure 2 illustrates crosslinked cartilage 50 having: (i) a butanediol bridge 90a between a hydroxyl group of a first GAG and a hydroxyl group of a second GAG; (ii) a butanediol bridge 90b between a hydroxyl group of a GAG and an amine group of a collagen fibril; and (iii) a butanediol bridge 90c between an amine group of a first collagen fibril and an amine group of a second collagen fibril. One will appreciate that in certain embodiments, reaction conditions can promote the formation of one or more specific type(s) of butanediol bridges 90. One will appreciate that, similar to the embodiment illustrated in Fig. 2A, one or more butanediol bridges between amine groups of the same collagen fibril and / or between hydroxyl groups of the same GAG molecule are also contemplated herein.

[0074] Fig. 2B also illustrates crosslinker 40. Crosslinker 40 can comprise a diol and / or an ether (e.g., 1,4 Butanediol diglycidyl ether or another reactive compound operable forgenerating one or more diol-containing bridges between molecules of cartilage 5). Some embodiments can include a combination of sulfone-containing bridges, diol-containing bridges (e.g., butanediol-bridges), and / or other carbon containing bridges, including bridges comprising a hydrocarbon diol having between 3 and 12 carbon atoms.

[0075] One will appreciate that the size, shape, and / or orientation, etc. of elements illustrated herein are not drawn to scale. Accordingly, such features should not be construed as limiting the scope of this disclosure or the invention described herein.

[0076] In some embodiments, the bridges comprise or occupy less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 10%, less than 5%, less than 2.5%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, between 0.000001% and 20%. between 0.001% and 15%, between 0.01% and 15%. between 0.01% and 10%, between 2.5% and 20%, between 0.01% and 2.5%, between 1% and 10%, and / or between 3% and 20% of the total number of potential bridges that could theoretically be introduced into the cartilage-derived implant based on the number of free hydroxyl and / or primary amines available to form bridges.

[0077] In at least one embodiment, the molecular bridge(s) present in the crosslinked cartilage (or cartilage-derived implant comprising crosslinked cartilage) provides resistance to enzymatic digestion. Accordingly, in some embodiments, the cartilage-derived implant is more resistant to enzymatic digestion than the source cartilage. As used herein, enzymatic “digestion’7and similar terms can include enzymatic resorption and / or degradation. As used herein, enzymatic degradation can refer to the breaking of molecular bonds by an enzyme. Similarly, enzymatic resorption can refer to the degradation and subsequent assimilation of a compound or tissue. One will appreciate that, where appropriate, reference to one or more of enzymatic digestion, degradation, and / or resorption can imply and / or include other forms of enzymatic activity, including but not limited to digestion, degradation, and / or resorption.

[0078] As used herein, “resistant to enzymatic activity,” “enzy matically resistant” and similar terms can refer to any statistically significant increase in resistance to or against enzymatic digestion compared to the source cartilage from which it was derived. In at least one embodiment, the cartilage-derived implant can exhibit a statistically significant (e.g., p<0.05) increase in stability against collagenase activity. One will appreciate, however, that other enzymes and / or forms of enzy matic activity' are contemplated herein, including forms of enzymatic digestion of cartilage or component(s) thereof.

[0079] As illustrated in Fig. 3 A, native, unprocessed cartilage can retain only about 21% of the original mass subjected to a collagenase treatment assay. However, in the illustratedembodiment, processed, stabilized, and / or crosslinked cartilage (i.e., a cartilage-derived implant) can retain at least, up to, greater than, or approximately 42% of the original mass (of the native, source cartilage) subjected to a collagenase treatment assay (p=0.01). Thus, processed cartilage can retain approximately twice the mass than can native, unprocessed cartilage when subjected to a collagenase treatment assay. Similarly, processed cartilage can retain approximately 20% more mass than can native cartilage when exposed to collagenase.

[0080] One will appreciate, however, that in other embodiments, processed cartilage can retain less than 2 times or greater than 2 times, 3 times, 4 times, 5 times, or 10 times more mass than can native cartilage when exposed to collagenase. Similarly, processed cartilage can retain greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. 80%. 85%. 90%. 95%. 98%, or 99% of the original mass when exposed to collagenase. Likewise, processed cartilage can retain greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% more of the original mass than can native cartilage when exposed to collagenase.

[0081] One will also appreciate that in certain embodiments more rigorous and / or milder processing conditions can result in increased resistance to enzymatic digestion compared to the source cartilage. Processing conditions, as known in the art and described further below, can include reagent concentration(s), processing time(s), processing temperature(s), processing pH, level of agitation, presence or absence of additional reagents or processing steps, number of repeats for one or more steps, and / or other variables. Exemplary embodiments of some illustrative processing conditions, protocol, and results are discussed in further detail below.

[0082] In certain embodiments, the molecular bridge(s) or crosslinking provides resistance to enzymatic digestion while not significantly altering one or more biomechanical properties (e.g., strength) of the cartilage-derived implant relative to the source cartilage. Without being bound to theory, the introduction of too many bridges can change the mechanical properties of a cartilage-derived implant. For instance, the number of molecular bridges can influence the biomechanical (compressive yield) strength of the cartilage-derived implant. The number of molecular bridges can also influence the compressibility, compressive yield stress, compressive yield strain at break, elasticity, feel. Young’s modulus, instantaneous stiffness, tensile strength, tensile strain, coefficient of friction, resilience, shock absorption, and / or other biomechanical properties of the cartilage-derived implant.

[0083] As illustrated in Fig. 3B. a processed, crosslinked, cartilage-derived implant can retain a comparable level of compressive yield strength (relative to the native, unprocessed, source cartilage). In the illustrated embodiment, native, non-crosslinked or non-stabilized,source cartilage has a compressive yield stress value of approximately 5.9 MPa (Std. Dev. = 1.6) and processed, crosslinked cartilage has a compressive yield stress value of approximately 6.0 MPa (Std. Dev. = 2.2). Thus, the compressive yield stress value of the processed, crosslinked cartilage falls within the range of the natural variation of the compressive yield stress value for the source cartilage or within a statistically relevant deviation or error value for the range of the natural variation of the compressive yield stress value for the source cartilage.

[0084] In some embodiments, the cartilage-derived implant can be resistant to cellular infdtration. For instance, upon implantation or assay conditions, the processed tissue can resist migration of cells from their sources of origin (e.g., during inflammations and / or cancerous growth). Without being bound to theory , cartilage and / or cartilaginous tissue is generally not conducive to cellular infiltration and is an inert structural material. Thus, in certain embodiments, the cartilage-derived implant can maintain the resistance to cellular infiltration present in native cartilage tissue. Furthermore, in some embodiments, the bridge(s) present in the cartilage-derived implant provide at least some protection from or resistance to or against molecular damage that can be caused by gamma or electron beam irradiation. Without being bound to theory, irradiation can weaken the biomechanical strength of cartilage and / or cartilaginous tissue (e.g., making the tissue less stiff by breaking molecular bonds). In at least one embodiment, crosslinking introduces new bonds that can help absorb the damage caused by irradiation. Thus, in certain embodiments, irradiated cartilage-derived implants can retain one or more biomechanical properties of the source cartilage (e.g., as a result of crosslinking).

[0085] In at least one embodiment, the cartilage-derived implant is processed in a manner that retains a uniform structure such as a structure that is consistent with the source cartilage material. For instance, a cartilage-derived material is rapidly frozen prior to being sterilized using radiation. The rapid freezing can be performed by a number of different means not limited to, but including submersion into liquid nitrogen, submersion into a mix of alcohol and dry ice, placement in one of the many varieties of commercially available blast chillers or freezers, exposure to liquid nitrogen sprays, direct contact with dry ice, or any similar method known to those skilled in the art of rapid freezing. Liquid nitrogen and solidified carbon dioxide (dry ice) are commonly used compressed gases for the cooling purposes, but other liquified or solidified gases can be used as well. In some embodiments, a cartilage-derived material can be rapidly frozen by placement in -80C freezer whereby the cartilage material is placed directly on a precooled metal pan or platform and a second precooled metal pan is placed on top of the cartilage material. In some embodiments the time to freeze a segment of cartilage-derivedmaterial is less than 70 seconds. 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, or less than 10 seconds.

[0086] Without being bound to theory, freezing a crosslinked cartilage-derived material (i.e., the crosslinked cartilage-derived material discussed hereinabove) by standard methods, such as placing the crosslinked cartilage-based material in a -20 C or -80 C freezer, can result in ice crystal formation. Because crosslinked cartilage has altered physical bonds compared to the source cartilage, these ice crystals can grow to create large, visible pores in the crosslinked cartilage-derived material. Upon irradiation of a frozen crosslinked cartilage derived material, both radiation-mediated physical crosslinking and any residual crosslinker or unreacted crosslinker pendant groups are activated to create new crosslinks within the frozen cartilage material. The new crosslinks formed during irradiation may cause the large pores formed by the ice crystals to remain in the cartilage material after thawing. For instance, the pores do not disappear upon thawing and the cartilage material does not re-expand to occupy the space created by the ice crystals. Cartilage material 200 with visible pores 201 after thawing are illustrated in FIG. 10. These large pores 201 render the material 200 non-uniform and may harm the structural properties and biocompatibility of the matenal 201. Thus, in certain embodiments, rapidly freezing a cartilage-based material can prevent pore formation or reduce the size of pores formed in the cartilage-derived implant compared to cartilage-derived implants frozen by standard methods, thereby preventing pore retention during irradiation. Furthermore, rapidly freezing the cartilage-derived material allows for the irradiation to eliminate unreacted ends of crosslinking groups without lengthy reaction and dilution times and provides an additional benefit of further stabilizing the cartilage in its desired state without large visible pores.

[0087] FIG. 11 A depicts aspects of an illustrative rapid freeze method, in which a cartilage- derived sample 300 is removed from its outer package (not shown) and placed in a mixture 302 of dry ice 304 and 91% isopropanol 306 for 3 minutes. Such a method may prevent formation of ice crystals in the cartilage-derived sample 300 thereby preventing the formation of pores in the cartilage-derived sample 300.

[0088] Figure 11B depicts aspects of an illustrative slow freeze method, in which a cartilage-derived sample (not shown) is wrapped in U” thick closed-cell foam insulation 400 and placed in -15°C freezer (not shown). Such a method may cause ice cry stals to form in the cartilage-derived sample which may form pores in the cartilage-derived sample (i.e., such as the pores illustrated in FIG. 10). Irradiation of the cartilage-derived sample while frozen may cause the cartilage-derived material to crosslink and retain the pores after the correspondingice crystals melt during thawing. As discussed above, pores may negatively effect the structural characteristics and / or biocompatibility of a cartilage-derived sample.

[0089] FIG.s 12A-12D illustrate cartilage-derived materials that have been frozen using different methods for purposes of comparison. FIG. 12A illustrates a sample of cartilage- derived material 500 that was slowly frozen surrounded by air at -15 °C. As illustrated in FIG 12A, visible pores 501 formed in the cartilage-derived material 500 as it was thawed. FIG. 12B illustrates a sample of cartilage-derived material 600 that was slowly frozen between two blocks of ice (not show n) at -15 °C. As illustrated in FIG. 12B, visible pores 601 formed in the cartilage-derived material 600 as it was thawed. FIG. 12C illustrates a sample of cartilage- derived material 700 that was slowly frozen surrounded by insulating material (not shown) at -15 °C. As illustrated in FIG. 12C, visible pores 701 formed in the cartilage-derived material 700 as it was thawed. FIG. 12D illustrates a sample of cartilage-derived material 800 that was rapidly frozen in dry ice and isopropanol (see FIG. 11 A) at about -80 °C. As illustrated in FIG. 12D, no visible pores formed in the cartilage-derived material 800 as it was thawed. However, it is apparent that after the cartilage-derived materials 500, 600, 700. and 800 were fully thawed (see the post thaw photo in each of FIG.s 12A-12D), there were no visible pores 501 any of the cartilage-derived materials 500-800. Notably, the cartilage-derived materials 500-800 were not irradiated in the frozen state. As discussed above, irradiation in a frozen state causes a cartilage- derived sample to crosslink and retain and pores formed while the cartilage-derived sample is frozen. Without being bound to theory, because the cartilage samples 500-800 were not irradiated in a frozen state, the pores 501 , 601 , 701 , and 801 that formed while freezing and were visible during thawing would not have healed if the cartilage-derived samples 500-800 were irradiated while frozen.

[0090] While pores in a cartilage-derived material may heal if the cartilage-derived material is not irradiated while frozen (as illustrated in FIG.s 12A-12D), photo-irradiation while a cartilage-derived material is thawed may damage and / or degrade the cartilage-derived material. Accordingly, preferred embodiments of the present invention include irradiation of a cartilage- derived material while the cartilage-derived material is in a frozen state.

[0091] FIG. 13A illustrates a sample of cartilage-derived matenal 900 that has been rapidly frozen rapidly frozen in dry ice and isopropanol (see FIG. 1 1A) at about -80 °C. The cartilage- derived material 900 was sterilized using irradiation while in a frozen state (i.e., before thawing) and was subsequently thawed. As illustrated in FIG. 13A, no visible pores are disposed in the cartilage-derived material 900 after thawing.

[0092] FIG. 13B illustrates a sample of cartilage-derived material 1000 that has been slowly frozen while surrounded by air at -15 °C. The cartilage-derived material 1000 was sterilized using irradiation while in a frozen state (i.e., before thawing) and was subsequently thawed. As illustrated in FIG. 13B, pores 1001 that formed during slow freezing remained in the cartilage derived material 1000 after thawing. While the cartilage-derived material 1000 was frozen while surrounded by air at -15 °C, other methods of slow freezing such as those described and illustrated above may cause also cause pores 1001 to form in the cartilage-derive material 1000.

[0093] In some embodiments, the cartilage-derived material is packaged in a single or double plastic pouch or other package and then rapidly frozen in its pouch. The package may act as a sterile barrier to the outside environment. In some embodiments the cartilage-derived implant is sterile with a sterility assurance level of at least, about, or less than 10’6, 10’5. 10’4, 10'3, or IO'2, where the sterility assurance level represents the probability of finding a non- sterile unit. In another embodiment, the cartilage-derived implant is produced from or by an aseptic process whereby the cartilage-derived implant possesses a sterility assurance level of IO’6, 10’5, IO4, 10’3, or IO’2.

[0094] Some embodiments include a method of processing animal-derived cartilage into a cartilage-derived implant (e.g., possessing one or more of the characteristics described above). Referring to Fig. 4, a method 100 can comprise a step 105 of obtaining a source cartilage and one or more optional steps, including: a step 110 of rinsing the cartilage (e.g., free or substantially free of blood and / or other component(s)); a step 115 of incubating the source cartilage in an anti pathogenic solution; a step 120 of treating the source cartilage with a galactose-alpha-l,3-galactose removal solution; a step 125 of treating the source cartilage with a detergent solution comprising: (i) a cationic detergent solution; (ii) one or more additional detergents (e.g., anionic, nonionic or zwitterionic); (iii) one or more ionic, nonionic, or zwitterionic surfactants; and / or one or more nucleases and / or enzymes; a step 130 of removing the detergent solution; a step 135 of treating the cartilage with a stabilizing agent that forms bridges or crosslinks between collagen and GAG molecules; a step 140 of rinsing the cartilage free of unreacted stabilizing agent; a step 145 of rinsing the processed cartilage-derived implant (e.g.. to adjust pH); a step 150 of packaging the cartilage-derived implant in a sealed package; a step 155 of rapidly freezing the cartilage-derived implant; a step 160 of sterilizing the packaged cartilage-derived implant; and a step 165 of thawing the cartilage-derived implant.

[0095] The source cartilage for the cartilage-derived implant can comprise septal, auricular, costal and / or articular cartilage from a pig, horse, cow, canine, human, or other animal. The animal can be a genetically modified animal that has been modified to exhibit certain geneticcharacteristics or advantages. For instance, in one embodiment, the source cartilage is derived from a non-primate animal genetically modified to lack or be deficient in galactose-alpha-1,3- galactose expression. In another embodiment, the source cartilage is derived from anon-human animal genetically modified to express one or more human proteins.

[0096] In one embodiment, the source cartilage can be recovered and / or cleaned (e g., free or substantially free of surrounding tissue) and placed in a vessel. The vessel, as known in the art. can comprise a conical tube, flask, beaker, or sterile container. Optionally, the source cartilage can then be rinsed with a water, salt solution, or a buffer solution (e g., to remove blood), illustratively for a period of 5 to 60 minutes and / or at a temperature between 1°C and 40°C with zero to three solution changes.

[0097] Optionally, the cartilage can then be incubated in an antipathogenic solution containing antibacterial, antifungal, and / or antiviral agents for a period of between 4 and 24 hours at a temperature between 20°C and 40°C with or without agitation. In some embodiments, the cartilage can then be incubated in a galactose-alpha-l,3-galactose removal solution containing a buffer (e.g. HEPES, phosphate, or Tris - having a pEI between 3 and 9) and a-galactosidase enzyme for a period of between 4 and 24 hours at a temperature between 1°C and 40°C with or without agitation. Other enzymes that digest galactose-alpha-1,3- galactose can be used in place of a-galactosidase, such as endo-galactosidase C. In at least one embodiment, treatment with the enzyme(s) does not significantly digest GAGs present in cartilage. In some embodiments, the a-galactosidase can comprise a purified enzyme and / or a recombinant protein. In some embodiments, the a-galactosidase can be present in a concentration betw een 0.1 U / mL and 10 U / mL. In some embodiments, the pH of the galactose- alpha-l,3-galactose removal solution can be between 3 and 9.

[0098] Following recovery and / or any of the aforementioned optional steps (i.e., cleaning, antipathogenic treatment, and galactose-alpha-l,3-galactose removal solution treatment), the source cartilage can be incubated in a solution containing a cationic detergent (referred to as the detergent solution). In one embodiment, the detergent solution can comprise a quaternary ammonium compound (QAC). In one embodiment, the specific QAC is myristyltrimethylammonium bromide (aka tetradecyltrimethylammonium bromide or TTAB). In other embodiments, a different cationic detergent can be used. Examples of other cationic detergents that can be used include cetyltrimethylammonium bromide, tridodecylmethylammonium chloride, dodecyltrimethylammonium bromide, hexadecylpyridinium chloride, cetylpyridinium chloride. orbenzyldodecyldimethylammonium bromide; however, it is understood that cationic detergents not listed here can also be used.

[0099] A combination of multiple cationic detergents can also be used in the detergent solution (rather than just one cationic detergent). Without being bound to theory, the detergent solution can be intended to kill any living cells and / or facilitate the removal of living cells and / or cellular remnants from the source cartilage. In some embodiments, cartilage can comprise a negatively charged tissue and, therefore, an oppositely charged detergent (e.g.. a cationic detergent) can more easily diffuse through the source cartilage compared to a detergent containing a negative charge (anionic), no charge (non-ionic), or a net zero charge (zwitterionic). Therefore, in certain embodiments, a cationic detergent can have or provide an improved capability to cleanse the source cartilage of living cells and / or cellular remnants, compared to other detergents.

[0100] In some embodiments, the cationic detergent is present in the detergent solution at a concentration at, near, or above the critical micelle concentration (CMC) for that specific detergent. In other embodiments, the cationic detergent is present at or near a concentration two to twenty times less than the CMC for the specific detergent. In some embodiments, the detergent solution comprises a detergent concentration greater than 0.001 pM and less than 25 mM.

[0101] Some embodiments include (the use of) a detergent solution comprising a nondetergent surfactant (e.g., in addition to the cationic detergent). In some embodiments, the surfactant aids in the removal of membrane associated proteins and / or galactose-alpha-1 ,3- galactose from the source cartilage. In at least one embodiment, the surfactant can be a nondetergent sulfobetaine (NDSB). In one aspect, the NDSB surfactant is 3-(l-Pyridino)-l- propane Sulfonate. In some embodiments, a different NDSB can be used, provided, and / or included. In some embodiments, the surfactant can facilitate the removal of cellular remnants.

[0102] Some embodiments include the addition of a nuclease to the cationic detergent solution. In some embodiments, the nuclease can comprise DNase and / or RNase, including combinations thereof. In other embodiments, the nuclease can comprise aSerratia marcescens derived nuclease such as Benzonase® or TurboNuclease. In other embodiments, the nuclease can comprise Cyanase™. One will appreciate, however, that generic, bio-equivalent, bioidentical, or structurally similar variations of such nucleases are also contemplated herein and / or can be included in certain embodiments. Without being bound to theory, the nuclease can be intended to facilitate the removal of RNA and / or DNA from the source cartilage. Some embodiments include the addition of antifungal agents to the detergent solution.

[0103] In some embodiments, the addition of antifungal agents to the detergent solution can reduce or eliminate the need for inclusion of a separate or additional anti-pathogenic solution treatment. One will also appreciate that other anti-microbial agents and treatments are contemplated herein.

[0104] In some embodiments, the detergent solution incubation step occurs with vigorous agitation. This agitation can induce a turbulent movement of detergent solution through or throughout at least a portion of the vessel. In some embodiments, the agitation occurs by vortexing, as known in the art. In other embodiments, the agitation can occur by shaking, rocking, and / or pressure mediated flow. The detergent solution incubation step can occur at a temperature between 20°C and 40°C, between 20°C and 30°C, or between 33°C and 40°C. In certain embodiments, a temperature closer to 40°C may reduce the necessary incubation time. In some embodiments, the detergent solution incubation step occurs for between 1 and 30 hours, between 2 and 18 hours, between 8 and 24 hours or between 18 and 24 hours.

[0105] As discussed in further detail below, in some embodiments, the cationic detergent can have or provide the advantage of more effectively removing galactose-alpha-l,3-galactose and / or MHC proteins from the source cartilage compared to other non-cationic detergent washes. In one or more embodiments, QAC detergents can be or comprise antiseptic compounds that eliminate or reduce the need for (additional) antimicrobial (i.e., antifungal, antibiotic, and / or antiviral) reagents or treatments in or during processing. The detergent solution can also have or provide the advantage of cleansing the source cartilage while not significantly altering one or more biomechanical properties (e.g., strength) of the cartilage- derived implant relative to the source cartilage. The detergent solution can also or alternatively have or provide the advantage of cleansing the cartilage while not removing vital biochemical components of cartilage (e.g., GAGs and / or type II collagen).

[0106] In some embodiments, cationic detergent treatment can substantially reduce the amount of source organism DNA associated with the cartilage compared to the native, unprocessed cartilage. In at least one embodiment, treatment with a cationic detergent can remove essentially all of the source organism DNA from the cartilage. In other embodiments, treatment with a cationic detergent can remove all but a trace amount of source organism DNA from the cartilage. Furthermore, in at least one embodiment, within a relevant concentration range, decreasing concentrations of TTAB are progressively more effective in reducing source organism DNA. Furthermore, certain concentrations of cationic detergent can remove up to 99% of source organism DNA. In some embodiments, treatment with a cationic detergent can remove up to, at least, greater than, or approximately 25%, 50%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of source organism DNA (or living cells and / or cellular remnants).

[0107] In certain embodiments, the process of removing an amount of source organism DNA can occur without (the need for) one or more (additional) DNase, RNase, and / or enzyme reagents.

[0108] In some embodiments, treatment with a cationic detergent does not significantly alter or affect one or more biomechanical properties (e.g., compressive strength) of the cartilage-derived implant relative to the source cartilage. In at least one embodiment, treatment with a cationic detergent does not significantly alter or affect one or more of the compressibility, compressive yield stress, compressive yield strain at break, elasticity, feel, Young’s modulus, instantaneous stiffness, tensile strength, tensile strain, coefficient of friction, resilience, shock absorption, and / or other biomechanical properties of the cartilage-derived implant. In at least one embodiment, treatment with a cationic detergent does not significantly alter or affect one or more biochemical properties (e g., molecular composition) of the cartilage-derived implant relative to the source cartilage. In at least one embodiment, even relatively high concentrations and / or relatively long treatment times of cationic detergents can cleanse the cartilage sample without significantly altering one or more biomechanical properties.

[0109] Without being bound to theory, the processing of source cartilage (e.g.. by treatment with one or more detergents, alcohols, or other reagents) can change the mechanical properties of a cartilage-derived implant. For instance, harsh detergents (e.g., anionic detergents, and some zwitterionic detergents) can influence the biomechanical (compressive yield) strength, compressibility, yield strain at break, elasticity, feel, Young's modulus, and / or other biomechanical properties of the cartilage-derived implant. In addition, alcohols can irreversibly dry and damage the cartilage by harming the molecular components and / or altering biochemical or biomechanical properties of the cartilage. Even mild detergents (e.g., non-ionic detergents, and some zwitterionic detergents) can damage cartilage with the prolonged exposure times and / or at high concentrations required to achieve the level of DNA (or living cells; cellular remnants; and / or galactose-alpha- 1,3 -galactose) reduction achieved in certain embodiments of the present invention.

[0110] In addition, anionic, zwitterionic, and non-ionic detergents may not remove living cells; cellular remnants; and / or galactose-alpha-l,3-galactose as effectively as one or more cationic detergents (e.g.. at one or more concentrations, for a given period of time, at a given temperature, with or without agitation, and / or under one or more other reaction conditions).However, in some embodiments of the present invention, one or more non-cationic detergents can be included in the detergent solution (e.g., at a concentration below the CMC of that detergent). The low concentration of non-cationic detergent can also facilitate the removal of certain cellular remnants in some embodiments of the present invention.

[0111] Following the detergent solution incubation, the source cartilage can be rinsed with a water, salt, or buffered salt solution in one to five successive steps or periods of between 15 and 240 minutes per period (e.g., at a temperature between 20 and 40°C with or without agitation). In some embodiments, a rinse step is performed to remove detergents and surfactants from the cartilage-derived implant.

[0112] In some embodiments, the cartilage-derived implant (e.g., having been treated and optionally rinsed free of the detergent solution) can be treated with a stabilization solution. The stabilization solution can comprise a multifunctional reactive molecule that is configured to and / or capable of reacting with one or more hydroxyl and / or primary amine groups. In at least one embodiment, the multifunctional reactive molecule is or comprises divinyl sulfone (e.g., where the vinyl groups can react with one or more hydroxyl groups to form one or more ether bonds and / or the vinyl groups can react with one or more primary amine groups to form one or more secondary amine bonds). In another embodiment, the multifunctional reactive molecule is or comprises a di- or tri-epoxide (e.g., where the epoxide groups can react with one or more hydroxyl groups to form one or more ether bonds and / or the epoxide groups can react with one or more primary amine groups to form one or more secondary amine bonds). In some embodiments, the multi-functional epoxide is or comprises 1 , 4 butanediol diglycidyl ether (BDDE). In other embodiments, the multi-functional epoxide is or comprises 1, 2,7,8- Diepoxyoctane. One will appreciate, however, that other multi-functional epoxides are contemplated herein.

[0113] In some embodiments, the concentration of the multifunctional reactive molecule, the pH of the solution, the temperature of the reaction, and / or the length of incubation time can all affect the rate at which the multifunctional reactive molecule reacts to form bridges between cartilaginous molecules (e.g., to form the cartilage-derived implant). Illustratively, if too many molecular bridges are introduced, the cartilage-denved implant may not possess the benefit of maintaining a comparable mechanical strength and feel compared to the source cartilage. For instance, too many molecular bridges can influence the biomechanical (compressive yield) strength of the cartilage-derived implant, making the implant more brittle and less elastic than native cartilage. Accordingly, in certain embodiments, stabilization solution treatment, the formation of molecular bridge(s), and / or crosslinking does not significantly alter one or morebiomechanical properties (e.g., strength) of the cartilage-derived implant relative to the source cartilage.

[0114] In some embodiments, treatment of the source cartilage with the stabilization solution occurs for a period of between 6 hours and 200 hours (e.g., at a temperature between 1°C and 30°C with or without agitation). In some embodiments the stabilization solution treatment step can be performed with agitation by vortexing, shaking, rocking, and / or pressure mediated flow.

[0115] In some embodiments the stabilization solution comprises a buffer that maintains the pH of the stabilization solution to a level greater than 9 and less than 12; or any value or range therebetween. In at least one embodiment, the pH of the reaction during treatment with stabilization solution can influence the number and / or type of molecular bridges formed and / or one or more mechanical properties of the resultant cartilage-derived implant. For instance, a processing pH value of 9 may result in a greater ratio of secondary amine bonds to ether bonds than a processing pH value of 12.

[0116] In some embodiments, the concentration of the multifunctional reactive molecule that reacts to form stabilizing bridges can be between 0. 1 mM and 100 mM, 1 mM and 20 mM, 0.5mM and lOmM or 20 mM and 40 mM. Different combinations of temperature, incubation time, pH and concentration (within the ranges stated herein) can result in a cartilage-derived implant with the benefit of resistance to enzymatic digestion and / or maintenance of the source cartilage mechanical feel and / or (compressive yield) strength. For instance, in some embodiments, a higher concentration with a shorter reaction time can achieve a similar end result as a lower concentration with a longer reaction time.

[0117] In at least one embodiment, lower concentrations of the multifunctional reactive molecule can be used to form fewer molecular bridges or crosslinks. In some embodiments, the fewer crosslinks introduced into the sample, the more natural feel and / or other properties the implant retains. Accordingly, in certain embodiments fewer than 20% of the total available bridge-forming sites are crosslinked during stabilization treatment. In other embodiments, the less than 20%, less than 19%, less than 18%, less than 17%. less than 16%, less than 15%, less than 10%, less than 5%. less than 2.5%, less than 1%. less than 0.1%, less than 0.01%, less than 0.001%, less than 0.0001%, between 0.000001% and 20%, between 0.001% and 15%, between 0.01% and 15%, between 0.01% and 10%, between 2.5% and 20%, between 0.01% and 2.5%, between 1% and 10%, and / or between 3% and 20% of the total number of potential bridges that could theoretically be introduced into the cartilage-derived implant based on thenumber of free hydroxyl and / or primary amines available to form bridges are crosslinked during stabilization treatment.

[0118] In some embodiments, after treatment with the stabilization solution, the cartilage- derived implant can be rinsed (e.g., free or substantially free from unreacted multifunctional reactive molecules; e.g., with or in a buffer that maintains the pH to a level greater than 9 and less than 12). In some embodiments the post stabilization rinse can comprise one to five successive rinse steps or periods (e.g.. of between 15 and 2000 minutes per period, at a temperature between 20°C and 30°C, and / or with or without agitation). In some embodiments, the post stabilization rinse permits or allows for a continuation of stabilization or bridge formation for reactive molecules in which only one end has reacted with a GAG or protein. In some embodiments, the stabilization rinse comprises a buffer that maintains the pH to a level greater than 9 and less than 12 and an additional primary amine-containing solute. In some embodiments, the solute comprises an amino acid (e.g., glycine). In some embodiments, the amine containing solute can react with a free reactive group that has not yet formed a bridge in the cartilage-derived implant.

[0119] In certain embodiments, the formation of one or more molecular bridges provides the cartilage-derived implant with resistance to enzymatic digestion. Accordingly, in some embodiments, the stabilization solution treatment makes the cartilage-derived implant resistant to enzymatic digestion. In at least one embodiment, stabilization solution treatment, the formation of molecular bridge(s), and / or crosslinking makes the cartilage-derived implant resistant to enzymatic digestion and does not significantly alter one or more biomechanical properties (e.g., strength) of the cartilage-derived implant relative to the source cartilage.

[0120] In some embodiments, treatment with the stabilization solution makes the cartilage- derived implant less susceptible to damage by electron beam and / or gamma radiation. One will appreciate that the stabilization solution treatment and / or high pH buffer rinse(s) can be performed or utilized on a cartilage-based implant that has not been first treated with the detergent solution described above. Thus, the stabilization solution treatment can comprise a standalone treatment or method of producing a cartilage-derived implant (e.g., that has been stabilized with molecular bridges, possesses the benefits of resistance to enzymatic resorption and / or protection from radiation-induced damage, and / or is mechanically comparable in feel and / or strength compared to the source cartilage material).

[0121] Following the optional high pH buffer rinse, the cartilage-derived implant can be rinsed with an isotonic saline and / or buffered saline solution (e.g., having a pH of between 5 and 8; or any value or range therebetween). The rinse step can comprise one to five successivesteps or periods of rinsing (e.g., between 15 - 240 minutes per period, at a temperature between 20°C and 30°C, and / or with or without agitation). With this step, the cartilage-derived implant is brought to a more physiologically relevant pH level.

[0122] In some embodiments, the cartilage-derived implant can be sealed in a package. In at least one embodiment, the package can safely withstand electron beam and / or gamma irradiation treatment. In some embodiments, the cartilage-derived implant can be packaged together with at least some residual solution. For instance, the cartilage-derived implant can be packaged together with less than 1 mL of solution. In some embodiments, the solution can comprise isotonic saline and / or buffered saline. In other embodiments the cartilage-derived implant can be sealed in a package with between 1 and 10 mL of additional solution. In other embodiments, the cartilage-derived implant is sealed in a package with no additional solution added. In at least one embodiment, the cartilage-derived implant can be packaged together with at least a trace amount of (or at least some residual) detergent (e.g., cationic detergent), surfactant, enzyme, multifunctional reactive molecule, and / or other reagent or component described herein.

[0123] In some embodiments, the packaged cartilage-derived material is rapidly frozen prior to being sterilized using irradiation. The rapid freezing can be performed by a number of different means not limited to, but including submersion into liquid nitrogen, submersion into a mix of alcohol and dry ice, placement in one of the many varieties of commercially available blast chillers or freezers, exposure to liquid nitrogen sprays, direct contact with dry ice, or any similar method known to those skilled in the art of rapid freezing. Liquid nitrogen and solidified carbon dioxide (dry ice) are commonly used compressed gasses for the cooling purposes, but other liquified or solidified gases can be used as well. In some embodiments, a cartilage- derived material can be rapidly frozen by placement in -80C freezer whereby the cartilage material is placed directly on a precooled metal pan or platform and a second precooled metal pan is placed on top of the cartilage material. In some embodiments the time to freeze a segment of cartilage-derived material is less than 70 seconds, 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, or less than 10 seconds.

[0124] In some embodiments, the frozen cartilage-derived implant can be sterilized by electron beam and / or gamma irradiation. Without being bound to theory, both forms of radiation sterilization are known to degrade or break the molecular bonds of the GAGs and / or collagen of cartilage, which can cause a change in the mechanical stiffness of the cartilage- derived implant. In at least one embodiment, electron beam irradiation can provide an advantage of requiring shorter exposure time compared to gamma irradiation. In anotherembodiment, electron beam irradiation can result in reduced degradation of GAGs and / or collagen, and / or reduced change in mechanical stiffness of the cartilage-derived implant. In another aspect (electron beam or gamma) sterilization can occur at a temperature between - 20°C and -80°C (e.g., about -40°C). In some embodiments, performing sterilization at a colder temperature can further reduce breakage of GAGs and further reduces changes of mechanical stiffness. In one embodiment the total dose of electron beam irradiation received by the implant can be between 1 and 20 kGy. In another embodiment, the total dose of electron beam irradiation received by the implant can be between 6 and 18 kGy.

[0125] In some embodiments, the cartilage-derived implant can be thawed after sterilization. The cartilage-derived implant, after thawing, does not contain visible pores or contains pores of reduced size when compared to cartilage-derived implants that were frozen by standard methods.

[0126] The invention also relates to methods of use for a cartilage-derived implant. In some embodiments, the cartilage-derived implant is used for repairing or reconstructing cartilaginous structures. In some embodiments, the method comprises obtaining a cartilage-derived implant (e.g., as described herein), sculpting the cartilage-derived implant into a specific shape, and / or implanting the cartilage-derived implant into the nose, ear, articular joint, or other anatomical location requiring repair with a cartilage-derived implant. In another embodiment, the method comprises obtaining the cartilage-derived implant, shaping the cartilage-derived implant to fit the shape of a cartilage defect, and / or implanting the cartilage-derived implant into the defect site. In one embodiment, a cartilage-derived implant whose source cartilage is septal cartilage can be shaped and / or implanted in the course of a rhinoplasty procedure.

[0127] In some embodiments, the cartilage-derived material can be made into various shapes as required for a given application. The shape can mean the outer 3D shape of the material or it could mean cut outs within a 3D shape. Cutouts could mean volumetric removals that protrude through the entire depth of the dimension being cut out, partial cutouts that do not encompass the entire depth of the dimension being cut out, fenestrations, perforations, or other manipulations. The cut-outs or other shape manipulations can be as small as a nanometer scale manipulation to a large size manipulation on the scale of the outer dimensions.

[0128] In some embodiments, the cartilage-derived material is manipulated into a particular shape or is given perforations, fenestrations, or cutouts before any treatments of the source cartilage. In other embodiments the for the cartilage-derived material is manipulated into a particular shape or is given perforations, fenestrations, or cutouts after any treatments of the source cartilage.

[0129] In some embodiments, it can be beneficial for the cartilage-derived material to be exposed to or rinsed in aqueous solutions such as water, normal saline, or pH-buffered solutions. In other embodiments, it can be beneficial for the cartilage-derived material to be exposed to or rinsed with more complex solutions such as platelet-rich plasma, blood, bone marrow aspirate, stem cell solutions, non-stem cell solutions, solutions containing pharmaceutical agents (e.g. anti-inflammatory drugs, antibiotics, pain relief agents, immunotherapies, etc.), and any combination thereof.

[0130] In some embodiments, the cartilage-derived material is a structure derived from a source of cartilage that is animal-derived or lab grown. In at least one embodiment, the source cartilage for the cartilage-derived material is derived from the septal, auricular, costal, intervertebral, tracheal, meniscal and / or articular cartilage of a shark, pig, horse, cow, canine, human, or other animal. In some embodiments, there are non-cartilage tissues attached to the source cartilage and not removed throughout the process resulting in a final cartilage-derived material with attached non-cartilaginous tissue. In another embodiment, the source cartilage for the cartilage-derived material is derived from lab grown cartilage. In some embodiments, there are non-cartilage tissues (lab grown or animal-derived) attached to the source cartilage and not removed throughout the process resulting in a cartilage-derived material with attached non-cartilaginous tissue. In other embodiments, the cartilage-derived material has non- cartilage tissue attached to the cartilage-derived material. For example, a cartilage-derived material can be attached to calcified or demineralized bone or bone-like tissue resulting in an osteochondral type structure. In other embodiments, the cartilage-derived material could be attached to metals, ceramics, or other biomaterials to create part-cartilage-derived material, part-biomaterial composite structures. In one embodiment, the cartilage-derived material is surrounded by lab grown or animal-derived non-cartilage extracellular matrix (ECM) structures such as smooth muscle or mucosal-like tissues to create a composite cartilage- derived material / non-cartilage ECM composite.

[0131] In one embodiment, the cartilage-derived material is used in medical applications. The medical applications of the cartilage-derived material include cartilage repair of any and / or all anatomical locations of cartilage. In other embodiments, the cartilage-derived material is used in medical applications where a cartilage or cartilage-like tissue is beneficial but not naturally present. In some embodiments the cartilage-derived material is utilized in the repair of nose, ear, trachea, articular surfaces, joint cartilage, intervertebral disc cartilage and all other cartilage-based or cartilage containing tissues. In some embodiments, the cartilage-derived material can be used to repair non-cartilaginous tissues. For example, the cartilage-derivedmaterial may be used as a spacer implant because of the naturally non-adherent nature of cartilage. In some applications, the increased compressibility and pliability of the cartilage- derived material relative to native cartilage will allow for easier handling and implantation of the cartilage-derived material.

[0132] Without being bound to theory, whereas other allograft derived materials can resorb over time, certain cartilage-derived implants described herein can provide resistance to enzymatic resorption. Likewise, whereas synthetic-derived materials used in rhinoplasty may extrude over time, some cartilage-derived implants described herein, being a natural material, can be resistant to extrusion. Likewise, whereas traditional costal cartilage grafts used in rhinoplasty may warp over time due to internal graft stresses, enzymatic resorption, or fibrous deposition, some cartilage-derived implants described herein can be resistant to enzymatic resorption and, by nature of being the same type of cartilage as the repaired, replaced, or augmented cartilage, can be less likely to elicit excess fibrous tissue deposition over time or experience unnatural stresses.

[0133] The following examples represent exemplary embodiments and are provided for illustrative purposes only. Accordingly, the disclosed examples are meant to illustrate one or more aspects of the invention and are not intended to limit the scope of the present invention. Example 1 : An exemplar7cartilage-derived implant

[0134] One embodiment comprising a cartilage-derived implant can be produced as follows. Septal cartilage from a pig is collected, dissected free of surrounding tissues, cut to dimensions of 2cm x 6cm, and then placed in a 50 mL container (e.g., tube) along with 45 mL of isotonic saline. The container is placed on a tube shaker and allowed to shake at 400 RPM for 15 minutes. After 15 minutes, the rinsate is removed from the container and replaced with 45 mL of various cationic detergent solutions (see Table 1 below). Detergents and surfactants used in these processes are listed below in Table 1 and include the following: Tetradecyltrimethylammonium bromide (TTAB), Cetyltrimethylammonium bromide (CTAB), 3-(l-Pyridino)-l -propane Sulfonate (NDSB 201), and Dimethylbenzylammonium Propane Sulfonate (NDSB 256). The container is then incubated at either 25°C or 37°C with high speed vortexing agitation for 24 hours. After 24 hours, the detergent solution is removed and replaced with 45 mL of isotonic saline. The container is incubated at 25°C with high speed vortexing agitation for five successive rinses of 1 hour each with the isotonic saline being changed between each rinse.Table 1Example 2: Stabilization treatment with a multifunctional reactive molecule

[0135] In one embodiment, a cartilage-derived implant produced in Example 1 is further processed with a stabilization solution to impart the benefits of stabilization. The cartilage- derived implant produced in Example 1 is placed in a 50 ml container with 45 mL of stabilization solution comprising 5 mM BDDE in a 100 mM bicarbonate buffer, pH 11. The container is placed on a tube shaker and allowed to shake at 300 RPM for 24 hours at 25 C. After 24 hours the stabilization solution is removed and replaced with 45 mL of 100 mM bicarbonate buffer pH 11. The container is incubated at 25°C with shaking at 1000 RPM for three successive rinses of 15 minutes, 1 hour, and 24 hours, respectively, with the buffer solution being changed between each rinse. Following the buffer rinses, the buffer is replaced with 45 mL of 50 mM Tris-buffered saline. pH 7.4. The container is incubated at 25°C with shaking at 300 RPM for three successive rinses of 15 minutes each with the Tris-buffered saline being changed between each rinse.Example 3: Freezing method determination

[0136] In one embodiment, a cartilage sample processed and crosslinked as described hereinabove without irradiation sterilization was subjected to four different freezing methods each with a different rate of freezing. In method 1, the sample was wrapped in 1 / 2-in closedcell foam and placed in a -15°C freezer (slowest freeze rate). In method 2, the sample was placed in the -15°C freezer with no insulation. In method 3, the sample was placed in the -15°C sandwiched betw een pre-conditioned Nordic Ice freezer blocks. In method 4, the sample wasrapidly submerged into a bucket of 91% isopropyl alcohol containing dry ice. After the device was frozen by each method, the device was thawed by running lukewarm water over the frozen sample. After 30 - 120 seconds, the surface of the device would thaw enough to make any pores visible through the plastic packaging. At that point, a picture was taken to record pore formation within the cartilage sample. Upon full thawing of the device, the ice crystals would melt completely, the pores would disappear, and the cartilage sample would return to looking like a solid material with no holes. The same cartilage sample was tested by all four methods. The sample when frozen by method 1 exhibited the most number of large pores that were presumably the result of ice crystal formation. The sample when frozen by Method 2 and Method 3 exhibited large pores, but not as many as Method 1. The sample when frozen by Methods 2 and 3 did not appear obviously different. The sample when frozen by Method 4 exhibited no pores. These results (the lack of pore formation by Method 4 and the significant pore formation seen in Method 1) demonstrate that the fastest freezing method prevents pore formation and support the conclusion that ice crystal formation known to occur during slow freezing methods is responsible for pore formation.Example 4: Irradiation Problem

[0137] In a second embodiment, 12 cartilage samples were processed and cross-linked as described hereinabove. These cartilage samples w ere frozen by placing the cartilage samples in a -80°C freezer. With freezing at -80°C, 4 devices exhibited minimal pore formation, 4 cartilage samples exhibited minor pore formation, and 4 cartilage samples exhibited significant pore formation. This data suggests that simple placement of cartilage samples in a -80°C freezer is not consistently capable of freezing fast enough to prevent pore formation although that may depend on placement within the freezer, whether freezer doors are frequently opened, and other variables that make this method of freezing unreliable. These 12 cartilage samples were then irradiated by electron beam in a container that maintains temperature at -40°C to ensure the cartilage samples remain frozen (6 at a dose of 18 kGy and 6 at a dose of 23 kGy). After irradiation, the cartilage samples were thaw ed. Rather than the ice cry stals melting and the pores disappearing, the pores remained in place. This demonstrates that without rapid freezing, the pores that formed as a result of ice crystal formation remain locked in place due to crosslinking that occurs during irradiation.Example 5: Rapid Freezing and Irradiation

[0138] In another example, 6 cartilage samples were processed and cross-linked as described hereinabove without irradiation sterilization. The samples were then frozen rapidly by Method 4 described above (submerging the sample in bucket containing isopropyl alcoholand dry ice). The devices were then irradiated by electron beam irradiation (17.5 kGy) with the devices being irradiated on a bed of dry ice to maintain a frozen state throughout irradiation. After irradiation, the samples were thawed and all samples were completely solid with no apparent pores or holes.Example 6: Rapid Freezing

[0139] In another example, 6 cartilage-derived material samples were processed and crosslinked as described previously (patents # US10016528B2 and US10814035B2). The samples were then frozen rapidly by Method 4 described above (submerging the sample in bucket containing isopropyl alcohol and dry ice). In this example the alcohol was allowed to reach a stable temperature of -25°C, -40°C, -55°C, or -72°C. Two samples were frozen by submersion in the alcohol at a temperature of either -25°C, -40°C. or -55°C and then the same two devices were thawed and frozen again by submersion with the alcohol at -72°C. At alcohol temperatures of -25°C, -40°C, and -55°C, visible pores were present in the cartilage material during thawing while at -72°C no visible pores were present in the cartilage during thawing.Example 7: Rapid Freezing

[0140] In another example, 23 cartilage-derived material samples were processed and crosslinked as described hereinabove. The samples were then frozen rapidly by submerging the sample in a bucket containing liquid nitrogen. During thawing, no visible pores were present in the cartilage during thawing.Example 8: Rapid Freezing and Irradiation

[0141] In another example, 72 cartilage-derived material samples were processed and crosslinked as described hereinabove. The samples were then frozen rapidly by submerging the sample in a bucket containing liquid nitrogen. The devices were then irradiated by electron beam irradiation (17.5 kGy) with the devices being irradiated on a bed of dry ice to maintain a frozen state throughout irradiation. After irradiation, all samples were completely solid with no apparent pores or holes.Example 9: Packaging and terminal sterilization

[0142] Another exemplary embodiment relates to terminal sterilization of the cartilage- derived implant. In this example, the cartilage-derived implant from Example I or 2 is placed in a plastic container and sealed to form a sterile barrier between the cartilage-derived implant and the outside environment. The cartilage-derived implant is then placed into a box containing dry ice and then transported to an electron beam facility, if necessary. The cartilage-derived implant is irradiated with one side of the graft against the dry ice and the other side in directexposure to the electron beam source. The cartilage-derived implant is then exposed to 8 - 12 kGy of irradiation.Example 10: DNA quantification assay

[0143] 100 mg samples were cut to fine pieces with a scalpel and digested in 1.5 rnL of 0.5 mg / rnL Proteinase K in 10X PBS with 5mM EDTA for 4 hr at 55°C with constant shaking. Sample digestions were then quantified using the Quant-iT™ High-Sensi tivi t DNA Assay Kit by Life Technologies (Carlsbad. CA).Example 11: DNA reduction in cationic detergent treated cartilage

[0144] As illustrated in Fig. 5, and corresponding Tables 2 and 3, vary ing concentrations of TTAB can remove varying amounts of source organism DNA. In summary, while native porcine septal cartilage contains greater than about 250 ng DNA per mg wet weight cartilage, cationic detergent treated cartilage-derived implants can comprise less than or between about 8.6 and about 25 ng DNA per mg wet weight cartilage. Similarly, Fig. 6, and corresponding Tables 2 and 3. illustrate percent reduction in source organism DNA following treatment with varying amounts of TTAB. In summary, within a relevant concentration range, decreasing concentrations of TTAB are progressively more effective in reducing source organism DNA. At lower concentrations, for instance, TTAB treatment effectively removes greater than 96% of source organism DNA from the cartilage-derived implant.

[0145] Table 2 displays the average values of percent DNA reduction and residual DNA amount for each of the tested concentrations of TTAB. Table 3 displays the replicate data for each tested concentration from which the averages were derived.Table 2Table 3Example 12: Galactose-alpha-l,3-galactose competitive ELISA

[0146] An ELISA plate was prepared by coating with 50 pL of 10 pg / mL BSA-alpha gal (Dextra Laboratories, UK) in TBS incubating overnight at 4 °C. The plate was then washed 3 times with PBS, blocked for 1 hr at 37 °C with 150 pL of 1.5% HSA, and then washed again3 times with PBS. 100 mg samples were minced and incubated in a 1: 100 M86 antibody (Enzo Life Sciences, Farmingdale, NY) solution in PBS overnight at 4 °C with slight shaking. Rabbit red blood cells known to possess 2 x 106alpha-gal epitopes per cell were also incubated in a 1: 100 antibody solution at various concentrations to create a standard curve. Samples and standards were centrifuged at 20,000g at 4 °C for 30 minutes and 50 pL of the supernatant for each sample and standard were loaded into wells of the prepared plate in triplicate. After a 2 hr room temperature incubation, the plate was washed and incubated for 1 hr with a hrp- conjugated goat anti-mouse antibody (1:500 in PBS). The plate was washed again, exposed for15 minutes with 50 pL of a TMB substrate and stopped with 50 pL of a 0.2M sulfuric acid solution. The absorbance of each well at 450 nm was measured by a plate reader.Example 13: Galactose-alpha- 1,3 -galactose reduction in cationic detergent treated cartilage

[0147] As illustrated in Fig. 7, and corresponding Table 4, cationic detergent can remove more galactose-alpha- 1,3 -galactose from the source cartilage than can zwitterionic detergent. In summary, while native source cartilage treated with 5 mg / mL CHAPS retains 1.281xl09galactose-alpha-l,3-galactose epitopes per mg wet weight cartilage, native source cartilage treated with 0.25 mg / mE TTAB retains only 4.97xl08galactose-alpha-l,3-galactose epitopes per mg wet weight cartilage. Thus, zwitterionic detergent treated cartilage retains greater than twice as much residual galactose-alpha-l,3-galactose than does cationic detergent treated cartilage. Similarly, cationic detergent treated cartilage can retain up to, greater than, or about 50% less galactose-alpha-l,3-galactose (or other immunogenic protein or effector) than does cartilage treated with zwitterionic detergent. Table 4 displays the values of galactose-alpha- 1,3-galactose epitopes remaining after detergent treatment at the indicated concentrations of CHAPS and TTAB, respectively.Table 4Example 14: Biomechanics assay

[0148] Two 6mm circular disks were obtained from each sample and the thickness of each disk was cut to ~4mm with a scalpel. After a tare load of 10N was placed on a disk, the disk was rapidly compressed in an unconfmed compression using an Instron Mini 55 and 500N load cell at a rate of 3mm / s to a stopping point of 85% compressive strain.Example 15: Retention of biomechanical strength in processed cartilage-derived implants

[0149] As illustrated in Fig. 8, and corresponding Tables 5 and 6, cartilage-derived implants retain a comparable level of biomechanical strength (relative to the native, unprocessed, source cartilage). In summary, the compressive yield stress values of crosslinked cartilage following treatment with various concentrations of the crosslinker BDDE falls within the range of the natural variation of the compressive yield stress values for the source cartilage and cartilage treated with TTAB only, or within a statistically relevant deviation or error value for the range of the natural variation of the compressive yield strength value for the source cartilage andcartilage treated with TTAB only. Lower concentrations of BDDE tend to provide average compressive yield stress values closer to those of the untreated and TTAB only samples. Thus, without being bound to theory, fewer molecular bridges between molecular components of the cartilage-derived implant may result in a more naturally feeling and functioning implant sample.

[0150] Table 5 displays the average values of compressive yield strength for native source cartilage, cationic detergent treated cartilage-derived implants, and crosslink stabilized cartilage-derived implants. Table 6 displays the replicate data for each tested concentration from which the averages in Table 5 were derived. Table 5Table 6Example 16: Enzyme digestion assay

[0151] 800 mg of each sample was rinsed 2 x 5 minutes in ultra-pure water. Samples were then cut in half and weights of each half were recorded. One half was incubated in 150 U / mL Type 1 A collagenase in a 50mM TES. 0.36 CaCb pH 7.4 buffer for 72 hr at 37°C followed by3 x 5 minute rinses in ultrapure water. Both halves were freeze-dried and percent mass remaining was calculated as the ratio between the digested and non-digested samples dry mass / wet mass values.Example 17: Enzymatic resistance of stabilized cartilage-derived implants

[0152] As illustrated in Fig. 9, and corresponding Tables 7 and 8, cartilage-derived implants can exhibit an increased resistance to enzymatic activity (e.g.. enzymatic digestion). In summary, the percent cartilage mass remaining after exposure to collagenase, progressively increases substantially logarithmically within a relevant range of increasing concentrations of the crosslinker BDDE. Lower concentrations confer less resistance and higher concentrations generally confer greater resistance (e g., to a maximum of greater than about 85-86% mass remaining) before leveling off. Accordingly, without being bound to theory, a greater number of molecular bridges between molecular components of the cartilage-derived implant may result in greater resistance against enzymatic activity.

[0153] Table 7 displays the average values of percent mass remaining for non-crosslinked cartilage-derived implants (TTAB only) and crosslink stabilized cartilage-derived implants following treatment with various concentrations of BDDE. Table 8 displays the replicate data for each tested concentration from which the averages in Table 7 were derived.Table 7Table 8Example 18: Unit conversions by reagent

[0154] In some embodiments, certain reagents are provided at a concentration having a defined unit of measurement. One will appreciate that unit conversions as known in the art and / or as provided herein are also contemplated. For instance, Table 9, below, displays exemplary unit conversions for the cationic detergent TTAB, the zwitterionic detergent CHAPS, and the crosslinking agent BDDE.Table 9

[0155] While various aspects and embodiments have been disclosed herein, other aspects and embodiments are contemplated. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting. It is noted that products, processes, compositions, kits, and methods according to certain embodiments of the present invention may include, incorporate, or otherwise comprise properties, features, components, members, and / or elements described in other embodiments described and / or disclosed herein. Thus, reference to a specific feature in relation to one embodiment should not be construed as being limited to applications only within said embodiment. In addition, various embodiments can be combined to form additional embodiments without departing from the scope of the invention or this disclosure.

[0156] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. While certain embodiments and details have been included herein and in the attached invention disclosure for purposes of illustrating the invention, it will be apparent to those skilled in the art that various changes in the products, processes, compositions, kits, and methods disclosed herein may be made without departing from the scope of the invention, which is defined in the appended claims. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. Various modifications that fall within the scope of the appended claims will be apparent to one skilled in the art.

Claims

CLAIMSWhat is claimed is:

1. A method for preparing a cartilage-derived material, comprising: obtaining treated cartilage derived from source cartilage, the treated cartilage comprising intact extra-cellular matrix having an increased resistance to enzy matic degradation by an extra-cellular matrix degrading enzyme as compared to the source cartilage; freezing the treated cartilage to form frozen, treated cartilage; and irradiating the frozen, treated cartilage while in a frozen state to form sterilized, frozen, treated cartilage.

2. The method of claim 1, wherein the source cartilage is derived from an animal or lab- grown source.

3. The method of claim 2, wherein the animal source cartilage comprises an explant from a pig, horse, cow, canine, human, or non-human animal and is selected from the group consisting of septal cartilage, auricular cartilage, costal cartilage, and articular cartilage.

4. The method of claim 1. wherein the step of freezing the treated cartilage involves submersion into liquid nitrogen, submersion into a mix of alcohol and dry ice, exposure to liquid nitrogen sprays, direct contact with dry ice, or placement in -80C freezer wherein the cartilage material is placed directly on a first precooled metal pan and a second precooled metal pan is placed on top of the cartilage material.

5. The method of claim 1. wherein the step of freezing is performed in less than 70 seconds, preferably less than 60 seconds, more preferably less than 50 seconds, still more preferably less than 40 seconds, still more preferably less than 30 seconds, still more preferably less than 20 seconds, still more preferably less than 10 seconds.

6. The method of claim 1, wherein the step of freezing the treated cartilage reduces the temperature of the treated cartilage from greater than 0 degrees Celsius to less than or equal to 0 degrees Celsius, or from greater than or equal to 4 degrees Celsius to less than or equal to 0 degrees Celsius, in less than 70 seconds, preferably less than 60 seconds, more preferably less than 50 seconds, still more preferably less than 40 seconds, still more preferably less than 30 seconds, still more preferably less than 20 seconds, still more preferably less than 10 seconds.

7. The method of claim 1, wherein the step of freezing the treated cartilage reduces the temperature of the treated cartilage to less than or equal to -72 degrees Celsius.

8. The method of claim 1, wherein: the sterilized, frozen, treated cartilage is devoid of visible pores or pores visible to the naked eye;the sterilized, frozen, treated cartilage does not exhibit visible pores or pores visible to the naked eye that are present in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed; and / or visible pores or pores visible to the naked eye in the sterilized, frozen, treated cartilage are of a reduced size as compared to the size of pores in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed.

9. The method of claim 1 further comprising thawing the sterilized, frozen, treated cartilage after irradiation to obtain an irradiated, enzyme-resistant cartilage material wherein: the irradiated, enzyme-resistant cartilage material is devoid of visible pores or pores visible to the naked eye; the irradiated, enzyme-resistant cartilage material does not exhibit visible pores or pores visible to the naked eye that are present in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed; and / or visible pores or pores visible to the naked eye in the irradiated, enzyme-resistant cartilage material are of a reduced size as compared to the size of pores in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed.

10. The method of claim 1, wherein the treated cartilage: has been treated with a cationic detergent; is devoid of amount of one or more components selected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins; and / or galactose-alpha-l,3-galactose; and / or has a reduced amount of amount of one or more components selected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins; and / or galactose-alpha-l,3-galactose, as compared to the source cartilage.

11. The method of claim 10, wherein the cationic detergent comprises one or more components selected from the group consisting of a quaternary ammonium compound, myristyltrimethylammonium bromide, cetyltrimethylammonium bromide,tridodecylmethylammonium chloride, dodecyltrimethylammonium bromide, hexadecylpyridinium chloride, cetylpyridinium chloride, benzyldodecyldimethylammonium bromide, and combinations thereof.

12. The method of claim 1, wherein the treated cartilage has been treated with at least one multifunctional reactive molecule configured to form one or more molecular bridges between molecules of the source cartilage, the one or more molecular bridges selected from the group consisting of: a hydrocarbon diol-containing bridge having between 3 and 12 carbon atoms; a sulfone-containing bridge; a molecular bridge between amine groups of one or more collagen fibrils; a molecular bridge between hydroxyl groups of one or more glycosaminoglycan molecules; and a molecular bridge between a hydroxyl group of a glycosaminoglycan molecule and an amine group of a collagen fibril, wherein the cartilage-derived implant is resistant to enzymatic digestion compared to the source cartilage.

13. The method of claim 12, wherein the at least one multifunctional reactive molecule is selected from the group consisting of: a molecule comprising more than one and less than 5 reactive epoxide groups; di vinyl sulfone; 1, 4 butanediol diglycidyl ether; 1, 2, 7. 8 diepoxy octane: and combinations thereof.

14. The method of claim 12, wherein the at least one multifunctional reactive molecule is provided in solution at a concentration of between 0. 1 mM and 100 mM.

15. The method of claim 1, wherein irradiating the frozen, treated cartilage while in a frozen state comprises exposing the cartilage-derived implant sealed in a radiation resistant package to at least one dose of electron beam radiation between 1 and 20 kGy and at a temperature of between -20°C and -80°C.

16. A cartilage derived material prepared by the method of any one of claims 1-15.

17. A method for preparing a cartilage-derived material, comprising: obtaining source cartilage from an animal or lab grown source, the source cartilage comprising intact extra-cellular matrix; treating the source cartilage to be more resistant to enzymatic degradation from an extracellular matrix degrading enzy me compared to the source cartilage without treatment and, optionally treating the source cartilage so as to remove an amount of one or more componentsselected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins; and galactose-alpha-l,3-galactose; freezing the treated cartilage at a rate that results in: an absence of visible pores or pores visible with the naked eye; an absence of visible pores or pores visible with the naked eye that are present in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed; and / or pores of a reduced size as compared to the size of pores in a cartilage material that has been frozen at a slower rate, frozen at a slower rate and subsequently irritated, frozen at a slower rate and subsequently thawed, and / or frozen at a slower rate, subsequently irritated, and then subsequently thawed; optionally irradiating the frozen, treated cartilage in the frozen state; and optionally thawing the irradiated, frozen, treated cartilage.

18. The method of claim 17 further comprising one or more steps selected from the group of steps consisting of: rinsing the source cartilage to remove cellular remnants; treating the source cartilage with a solution comprising a cationic detergent; rinsing the source cartilage to remove at least some cationic detergent; rinsing the cartilage-derived implant with a solution comprising a primary amine-containing solute; sealing the cartilage-derived implant in a package; sealing the cartilage-derived implant in a radiation resistant package; sterilizing the cartilage-derived implant sealed in a radiation resistant package by irradiation; seeding the cartilage-derived implant with living human cells; sculpting the cartilage-derived implant; and implanting the cartilage-derived implant into a subject.

19. A cartilage-derived material, comprising: intact extra-cellular matrix of a source cartilage, optionally exhibiting an increased resistance to enzy matic degradation by an extracellular matrix degrading enzyme as compared to the source cartilage; and at least one of: sterility gained by irradiating while in a frozen state;an absence of visible pores or pores visible to the naked eye.

20. The cartilage-derived material of claim 19, wherein the material, relative to the source cartilage, comprises a reduced amount of one or more components selected from the group consisting of: living cells; cellular remnants; blood; DNA; major histocompatibility complex proteins; and galactose-alpha-l,3-galactose.

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