Amnion patch and uses thereof

Abstract

Provided herein is a decellularized amnion-based article and uses thereof. In particular, provided herein is a decellularized amnion patch and uses thereof, for example, in tissue regeneration and repair.

Description

AMNION PATCH AND USES THEREOFGOVERNMENT SUPPORT

[0001] This invention was made with government support under AR079114 awarded by the National Institutes of Health, and 1332329 awarded by the National Science Foundation. The government has certain rights in the invention.RELATED APPLICATIONS

[0002] This application claims priority to provisional patent application 63 / 573,034, filed April 2, 2024, which is herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] Provided herein is a decellularized amnion-based article and uses thereof. In particular, provided herein is a decellularized amnion patch and uses thereof, for example, in tissue regeneration and repair.BACKGROUND

[0004] An estimated 528 million individuals globally are affected by osteoarthritis (OA). Observed trends in osteoarthritis have revealed a significant increase in prevalence, with a 113% rise in diagnosed cases since 1990. The knee joint is identified as the most commonly impacted site in osteoarthritis.

[0005] Symptoms of OA include joint pain that often worsens during or after movement, stiffness that is most noticeable upon waking or after being inactive, tenderness or inflammation, loss of mobility, a grating or crackling sensation in the joint, bone spurs, and joint deformity.

[0006] The current management strategies for OA predominantly rely on non-disease modifying interventions. These conventional treatments include non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroid injections, and viscosupplementation (lubrication injections), primarily aiming at symptom relief rather than addressing the underlying causes of OA. While these approaches may provide temporary relief, they do not effectively halt disease progression or promote tissue repair. Consequently, patients often face the prospect of more invasive surgical interventions, such as total joint replacements, which carry heightened risks (e.g., infection, implant failure) and prolonged recovery periods.

[0007] This underscores a critical gap in current treatment modalities, highlighting the urgent need for innovative therapeutic options that not only alleviate symptoms but also stimulate tissue regeneration and have the potential to modify the disease course.SUMMARY

[0008] Provided herein is a decellularized amnion-based article and uses thereof. In particular, provided herein is a decellularized amnion patch and uses thereof, for example, in tissue regeneration and repair.

[0009] The compositions and methods described herein meet an unmet need for non-invasively treating symptoms of OA or other joint disorders, while promoting healing and tissue regeneration.

[0010] For example, in some embodiments, provided herein is a decellularized amnion membrane article. The present disclosure is not limited to a particular format of the article. Any number of non-micronized configurations are contemplated including but not limited to a patch or a sheet. In some aspects, the article has a DNA level below 50 ng / mg of dry tissue.

[0011] In some embodiments, the article is lyophilized (e.g., for storage and / or transport).In certain aspects, the article is twisted around an absorbable material (e.g., yam).

[0012] The articles of the disclosure may be decellularized using any suitable method. In one aspect, the decellularization comprises the use of a base (e.g., sodium hydroxide) and mechanical scraping (e.g., with surgical gauze or other suitable material). In some embodiments, the article is fiirther processed by lyophilization and / or sterilization (e.g., UV sterilization).

[0013] The present disclosure is not limited to the source of the amnion membrane. In some embodiments, the amnion membrane is isolated from a subject (e.g., a human or non-human mammal). In some embodiments, the amnion membrane is isolated from a placenta.

[0014] Also provided is a composition, kit, or system comprising an article described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition, kit, or system fiirther comprises a delivery device (e.g., comprising one or more of a plurality of syringe ports, a plurality of stoppers, a delivery needle, or a plunger). In some aspects, the delivery needle is configured to deliver an article to a specific location in a subject. In some embodiments, the article is preloaded into the delivery device.

[0015] In some aspects, the composition further comprises an active agent (e.g., absorbed into the article).

[0016] Further provided is a delivery device comprising an article described herein. In some embodiments, the article is pre-loaded into the delivery device. In some embodiments, thedelivery device further comprises one or more of an inert fluid (e.g., saline or buffer), an additional therapeutic agent, a scaffold, or a bioactive fluid.

[0017] Additional embodiments provide an amnion structure, comprising: a plurality of decellularized amnion membrane articles, wherein each article comprises a plurality of edges, and wherein at least two of the edges of the articles are sealed to each other. In certain aspects, at least one of the edges of the articles are not sealed. For example, in some embodiments, the sealed edges form a bag-like structure.

[0018] In certain aspects, the structure is filled with or coated with an active agent. The present disclosure is not limited to particular active agents. Examples include but are not limited to amniotic fluid, synovial fluid, fat, hyaluronic acid, cells, stem cells, small molecule pharmaceuticals, lubricants, placental fragment, decellularized extracellular matrix, degradable fabric, or electrospun mesh. Examples of lubricants include but are not limited to, glycerol, PEG, a phospholipids, a poloxamers, a fatty acids, a silicone-based lubricant, polyacrylamide, or a liposome-based lubricant.

[0019] Also provided is a method of generating a decellularized amnion membrane article, comprising a) obtaining an amnion membrane; b) contacting the amnion membrane with a base; and c) mechanically scraping the membrane. In some embodiments, the base is sodium hydroxide.

[0020] Certain aspects of the disclosure provide a method of treating osteoarthritis, comprising delivering an article described herein to a joint of a subject in need thereof. The present disclosure is not limited to particular joints. Examples include but are not limited to a knee, elbow, spinal vertebrae / disk spaces, wrist, toe, finger, shoulder, or hip joint. In some embodiments, the article is delivered to the joint via injection (e.g., using a delivery device) surgical implantation, or fixation. In some embodiments, the delivery device further performs joint aspiration (e.g., during the same procedure as the delivery of the article). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human subject. In some embodiments, the subject is a dog, a cat, a horse, cattle, or a pig. In some embodiments, the article preserves or regenerates articular cartilage in the joint and / or induces upregulation of lubricin in the joint.

[0021] Other aspects provide a method, comprising delivering an article described herein to a subject in need thereof. In some embodiments, the article is delivered to a joint, a tissue (e.g.,muscle), bone, a wound, an eye, or a tooth of the subject.

[0022] Additional embodiments are described herein.DRAWINGS

[0023] FIG. 1 shows where aspects of surgical implantation of placental tissue.

[0024] FIG. 2 is a graph with data for collagen quantification of decellularized and control amnion patches.

[0025] FIG. 3 is a graph with data for comparative quantification of total DNA in decellularized and control groups.

[0026] FIG. 4 shows propidium iodide staining for DNA in control vs NaOH treatment groups.

[0027] FIG. 5 shows safianin O staining that demonstrates that decellularized amnion patches preserve hyaline cartilage in OA.

[0028] FIG. 6 shows an exemplary delivery device of embodiments of the present disclosure.

[0029] FIG. 7 shows a schematic of in vitro testing of decellularized amnion patches using rat primary cells.

[0030] FIG. 8 shows a schematic of a study of the use of decellularized amnion patches in an animal model of OA.

[0031] FIG. 9A-B shows lubricin staining of animal treated with decellularized amnion patches and control animals.

[0032] FIG. 10 shows a flow chart of an exemplary method of obtaining decellularized amnion patches of embodiments of the disclosure.

[0033] FIG. 11 shows photographs of A) decellularized amnion membrane and cut amnion pieces. B) Pieces of amnion inside the folded membrane. C) & D) A pillow-like structure formed by crosslinking three sides of the decellularized amnion membrane.

[0034] FIG. 12 shows a schematic and images for an exemplary method of twisting an amnion membrane using an absorbable yam to generate a tightly rolled decellularized amnion membrane.

[0035] FIG. 13 shows an exemplary amnion structure with sacrificial and highly absorbent components.DETAILED DESCRIPTION

[0036] Before the disclosed processes and materials are described, it is to be understood that theaspects described herein are not limited to specific embodiments, or examples, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0037] Osteoarthritis (OA) is characterized by bond spurs, cartilage loss, and joint space narrowing. At the cellular level, joints with OA exhibit high levels of proinflammatory cytokines and altered gene expression levels relative to healthy joints.

[0038] Current non-disease modifying interventions and invasive surgical procedures are limited in either not addressing the underlying causes of OA or by requiring invasive procedures with long recovery times. Additionally, in current clinical practice, aspirations and injections of synovial / articular joints typically occur as separate procedures. Multiple injections into the knee joint increase the risk of complications such as infection, tissue damage, and nerve injury.

[0039] Provided herein are decellularized amnion articles (e.g., patches) that overcome limitations of existing treatments for OA. The compositions and methods described herein address the underlying causes of OA and other joint disorders by promoting healing and regeneration while providing symptom relief. The compositions and methods described herein further, in some aspects, integrate aspiration and injection functionalities into a single procedure, minimizing the number of incisions and injections a patient undergoes.

[0040] The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, the described technologies are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0042] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. By way of example, "anelement" means one element or more than one element. Similarly, the adjective “another,” when used to introduce an element, is intended to mean one or more elements. The terms “including” and “having” are intended to be inclusive such that there may be additional elements other than the listed elements. The term “exemplary” is not intended to be construed as a superlative example but merely one of many possible examples.

[0043] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0044] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0045] The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.

[0046] The terms “about” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ± 10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure as used herein.

[0047] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0048] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0049] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionallyincluding more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0050] The phrase "one or more," as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0051] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as being modified in all instances by the term "about," meaning within 10% of the indicated number (e.g., "about 10%" means 9%-l 1% and "about 2%" means 1.8%-2.2%).

[0052] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Generally, unless otherwise expressly stated herein, "weight" or "amount" as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising the ingredient, wherein the raw material may be described herein to comprise less than and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.

[0053] All ranges and amounts given herein are intended to include subranges and amounts using any disclosed point as an end point. Thus, a range of "1% to 10%, such as 2% to 8%, such as 3% to 5%," is intended to encompass ranges of "1% to 8%," "1% to 5%," "2% to 10%, " and so on. All numbers, amounts, ranges, etc., are intended to be modified by the term "about," whether or not so expressly stated. Similarly, a range given of "about 1% to 10%" is intended to have the term "about" modifying both the 1% and the 10% endpoints. Further, it is understood that when an amount of a component is given, it is intended to signify the amount of the active material unless otherwise specifically stated.

[0054] As used herein, the term “administering” means the actual physical introduction of a composition into or onto (as appropriate) a subject, a host, or cell. Any and all methods of introducing the composition into the subject, host or cell are contemplated according to the disclosure; the method is not dependent on any particular means of introduction and is not to beso construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein. “Providing” means giving, administering, selling, distributing, transferring (for profit or not), manufacturing, compounding, or dispensing.

[0055] The term “composition” as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such a term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing an agent (e.g., decellularized amnion article) of the present disclosure and a pharmaceutically acceptable carrier and / or excipient. When an agent of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the agent of the present disclosure is contemplated. Accordingly, the pharmaceutical compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to an agent of the present disclosure. The weight ratio of the agent of the present disclosure to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Combinations of an agent of the present disclosure and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used. In such combinations the agent of the present disclosure and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

[0056] As used herein, the term “treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., OA). A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease orsymptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease.

[0057] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0058] The term “subject” or “patient” is used herein to refer to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g. , a duck or a goose), and a shark. In an embodiment, the subject or patient is a human subject or a human patient, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In one embodiment, the subject is in the prenatal period, newborn to one year of age, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95- 100 years of age. Values and ranges intermediate to the above recited ranges are also intended to be part of this disclosure. In addition, ranges of values using a combination of any of the aboverecited values as upper and / or lower limits are intended to be included. As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0059] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

[0060] A significant change is any detectable change that is statistically significant in a standard parametric test of statistical significance such as Student’s t-test, where p < 0.05.

[0061] All statements herein reciting principles, aspects, and embodiments of the disclosure, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure.

[0062] Various other components may be included and called upon for providing for aspects of the teachings herein. For example, additional materials, combinations of materials and / or omission of materials may be used to provide for added embodiments that are within the scope of the teachings herein. Adequacy of any particular element for practice of the teachings herein is to be judged from the perspective of a designer, manufacturer, seller, user, system operator or other similarly interested party, and such limitations are to be perceived according to the standards of the interested party.

[0063] In the disclosure hereof any element expressed as a means for performing a specified function is intended to encompass any way of performing that function including, for example, a) a combination of circuit elements and associated hardware which perform that function or b) software in any form, including, therefore, firmware, microcode or the like as set forth herein, combined with appropriate circuitry for executing that software to perform the function.Applicants thus regard any means which can provide those functionalities as equivalent to those shown herein. No functional language used in claims appended herein is to be construed as invoking 35 U.S.C. §112( / ) interpretations as “means-plus-function” language unless specifically expressed as such by use of the words “means for” or “steps for” within the respective claim.Decellularized amnion articles

[0064] In an aspect, disclosed herein is a decellularized amnion article. The amnion (or amniotic) membrane is the innermost layer of the placenta and is one layer of the amniotic sac that surrounds a fetus in utero (See e.g., Gude N.M., Roberts C.T., Kalionis B., King R.G.Growth and function of the normal human placenta. Thromb. Res. 2004; 114:397-407. doi: 10.1016 / j.thromres.2004.06.038). The human amniotic membrane (hAM) is sourced from the inner and outer layers of the amniotic sac and comprises two distinct, connected membranes:amnion and chorion. The smooth inner amnion membrane is a thin, tensile, avascular, semitransparent structure without nerve innervation, muscle, or lymphatic vessels, with a reported relative thickness of about 20-500 pm or about 35-60 pm. It comprises three major histological structures: an epithelial monolayer, a thick basement membrane, and an avascular stroma in contact with the underlying chorion.

[0065] The present disclosure is not limited to the source of the amnion membrane. In some embodiments, the amnion (or amniotic) membrane is isolated from a subject (e.g., a human or non-human mammal such as a dog, cat, pig, or cattle). In some embodiments, the amnion membrane is isolated from placental tissue (e.g., obtained from a mammalian subject after the subject has given birth, for example via cesarean section). Examples of suitable placental tissues include but are not limited to, the amniotic sac (e.g., amnion and / or chorion) and the umbilical cord.

[0066] The present disclosure is not limited to a particular format of the article. Any number of non-micronized configurations are contemplated including but not limited to a patch or a sheet. Any suitable format may be utilized. The size and shape of the article can be selected based on the specific application or location of use.

[0067] The articles of the disclosure may be decellularized using any suitable method. In one aspect, the decellularization comprises the use of a base (e.g., sodium hydroxide) and mechanical scraping (e.g., with surgical gauze or other suitable material), although any suitable method may be utilized. Any suitable base such as sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium oxide, sodium carbonate, sodium bicarbonate, ammonium carbonate, or any combinations thereof can be used for the decellularization. In some embodiments, the base is sodium hydroxide. For example, in some embodiments, the decellularization method comprises the steps of a) obtaining an amnion membrane; b) contacting the amnion membrane with a base such as sodium hydroxide; and c) mechanically scraping the membrane.

[0068] In some embodiments, after decellularization, the article is lyophilized and / or sterilized. The article can be fabricated into any suitable shape (e.g., patch or sheet), depending on the end use. In some embodiments, articles are lyophilized and / or sterilized before and / or after fabrication.

[0069] In some embodiments, the DNA content of decellularized amnion membranes is determined in order to confirm that sufficient cells have been removed from the membrane. For example, in some aspects, the article has a DNA level below about 50 ng / mg of dry tissue (e.g., below 50 ng / mg, below 40 ng / mg, below 30 ng / mg, or below 20 ng / mg). In some embodiments, the article has a DNA level of about 10-50 ng / mg. In some embodiments, the article has a DNA level of about 20-50 ng / mg. In some embodiments, the article has a DNA level of about 30-50 ng / mg. In some embodiments, the article has a DNA level of about 10-50 ng / mg. In some embodiments, the article has a DNA level of about 40-50 ng / mg. In some embodiments, the article has a DNA level of about 25-50 ng / mg.Compositions and devices

[0070] In some embodiments, the article is provided as a component of a composition, kit, or system. In some embodiments, the composition is a pharmaceutical composition.

[0071] Depending on the target sought to be altered by treatment, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include intramuscular, intra-articular, subcutaneous, intramedullary, intrathecal, local injection, or surgical administration.

[0072] For injection, the pharmaceutical compositions may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0073] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active agents (e.g., decellularized amnion article) in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0074] Compositions comprising an agent of the disclosure formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0075] The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be local (e.g., to a joint via injection or surgical incision), or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0076] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, earner compounds and other pharmaceutically acceptable carriers or excipients.

[0077] Thus, in some embodiments, pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0078] The pharmaceutical formulations, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers.

[0079] Thus, in some embodiments, the compositions may be formulated into any of many possible dosage forms such as, but not limited to, as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0080] The pharmaceutical compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions, such as preservatives and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the agents. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the active agent (e.g., decellularized amnion article) of the formulation.

[0081] In some embodiments, a delivery device is provided. Exemplary devices are shown in FIG. 6. For example, in some embodiments, the device comprises one or more of a plurality of syringe ports 2, a plurality of stoppers 3, a delivery needle 4, or a plunger 1. The syringe ports provide access to the device body 5 for introducing an article described herein (e.g., via a syringe), along with any additional agents or carriers. The stoppers 3 serve to contain the article and any additional agents inside the device. The needle 4 is used, for example, to deliver the article and any additional agents to a joint or other location within the body. Likewise, the plunger 1 serves to aid in delivery of articles. In use, the plunger 1 can be withdrawn during loading of the device body 5 (e.g., via the syringe port(s)) and then once the needle 4 is inserted into the delivery location, the plunger 1 can be depressed to ensure delivery of the article.

[0082] In some aspects, the delivery device is provided as a component of a kit or system. In some embodiments, the article is preloaded into the delivery device. In some embodiments, the device and the article are provided separately and the end user loads the device with the article.

[0083] In certain aspects, kits and / or systems comprise lyophilized articles. In some embodiments, devices are pre-loaded with lyophilized articles. Prior to use, the article can be rehydrated (e.g., in the device or outside of the device), using any suitable liquid (e.g., buffer or saline).

[0084] In some embodiments, prior to being loaded into a delivery device, articles are twisted or otherwise condensed around an absorbable material (e.g., yam). One exemplary method for generating a twisted article is shown in FIG. 12. This generates a tightly rolled or condensed article that can be inserted into a needed or other delivery device.

[0085] In some embodiments, the disclosure provides an amnion structure, comprising: a plurality of decellularized amnion membrane articles or a single folded article, wherein each article comprises a plurality of edges, and wherein at least two of the edges of the articles are sealed to each other. In certain aspects, at least one of the edges of the articles are not sealed. For example, in some embodiments, the sealed edges form a bag-like structure.

[0086] One exemplary embodiment of such pillow or bag-like structures is shown in FIG. 11 A- D. As shown in FIG. 11 A, two pieces of decellularized amnion membrane or a single folded piece of membrane serve as the outside of the structure. Before or after filling, the edges or sides of the membrane pieces are sealed (e.g., via crosslinking) to form a pillow or bag structure.

[0087] In some embodiments, the pillow structures serve as a cushion (e.g., inside a joint). For example, in some embodiments, the pillow structure serves as a cushion to reduce pressure between two surfaces where mechanical stress contributes to a problem such as osteoarthritis or shoulder impingement. Additionally, it can fimction as a biologic delivery system, gradually releasing therapeutic factors or lubricants from both the amnion sheet and the materials enclosed within it.

[0088] In certain aspects, the structure is filled with or coated with an active agent. The present disclosure is not limited to particular active agents. Examples include but are not limited to amniotic fluid (e.g., allogenic or xenogenic), synovial fluid, fat (e.g., processed autologous, allogenic, or xenogenic fat), hyaluronic acid, cells, stem cells, small molecule pharmaceuticals, lubricants, placental fragments (e.g., amnion, chorion, or umbilical cord fragments), decellularized extracellular matrix (ECM, e.g., cartilage or bone), hydrogels, degradable fabric, or electrospun mesh.

[0089] In some aspects, the structures are used to improve lubrication, which can be done by incorporating lubricating agents into the structure. Examples of lubricants include but are not limited to, glycerol, PEG, a phospholipids, a poloxamers, a fatty acids, a silicone-based lubricant, polyacrylamide, Pluronic, or a liposome-based lubricant.

[0090] In certain embodiments, the structure is filled with an active agent as shown in FIG. 13. FIG. 13 shows a structure comprising a decellularized amnion membrane covering and sealed around a sacrificial material or highly absorbent substance.

[0091] In some aspects structures such as those shown in FIG. 13 are filled with an active agent. In certain embodiments, the active agent is in a solid form (for example, apiece of cartilageECM, collagen membrane, degradable polymeric membranes, micronized tissue, or other suitable solid materials) or in a fluid form like hydrogel (for example, ECM hydrogel, Hyaluronic acid, or other suitable fluid materials) to provide cushioning.

[0092] In an embodiment, the structure serves as an artificial placenta in the joint by using amniotic fluid (or Synovial fluid) inside and amniotic membrane outside. In this way, amniotic fluid works as a shock absorbent and improves cushioning and the decellularized amniotic membrane works as a protective layer, and both release biological factors.

[0093] In some embodiments (e.g., those shown in FIG. 13), the device incorporates fluid-based materials into the amnion patch system using sacrificial or highly absorbent components. This component (sacrificial or highly absorbent) is completely covered by amnion and the device is going to be sealed (with heat, suture, a knot, or any other suitable sealing method) to make sure there is no leakage. Then, a fluid-form material (such as amniotic fluid, hydrogels, glycerol, or any other suitable fluid-form material) is injected into that component and then the desired fluid is placed into the device.

[0094] In an embodiment, the sacrificial or highly absorbent components is removed prior to transplantation. In another embodiment, the device is transplanted with the sacrificial or highly absorbent components. If the device is transplanted with the sacrificial or highly absorbent components, in some aspects, it also comprises drug-releasing agents in its structure to release drugs (e.g., pain reliever drugs, modulators of inflammation (e.g., anti-inflammatory agents), healing, or regeneration) in a short amount of time or over a long time period (e.g., extended release).

[0095] Kits and systems described herein may further comprise comprises one or more additional components, for example, an inert fluid (e.g., saline or buffer), an additional therapeutic agent, a scaffold, a tissue extracellular matrix, or a bioactive fluid. In some embodiments, the additional agents are preloaded into a device. In other aspects, the additional agents are provided separately (e.g., in separate containers).Methods of treatment

[0096] The decellularized amnion articles described herein find use in a variety of research, screening, and clinical applications. Exemplary methods are described herein.

[0097] As described herein, the articles of the disclosure further promote biomolecule (e.g., growth factor or cytokine) stimulation and induce upregulation of lubricating proteins such aslubricin and hyaluronic acid or inflammatory or regenerative modulators. Consequently, in some aspects, the decellularized amnion articles described herein promote tissue regeneration and facilitate cartilage regeneration and / or repair (e.g., by acting as a cushion in the joint space or by protecting the tissue withing the joint or by releasing inflammatory or regenerative modulators). The articles further find use in preventing degenerative musculoskeletal disorders.

[0098] Certain aspects of the disclosure provide a method of treating and / or preventing osteoarthritis in a subject, comprising delivering an article described herein to a joint of a subject in need thereof.

[0099] The present disclosure is not limited to particular joints. Examples include but are not limited to a knee, toe, finger, shoulder, or hip joint.

[0100] In some embodiments, the article is delivered to the joint via injection using a delivery device. While exemplary delivery devices are described herein, the disclosure is not limited to a particular delivery device or method. In some embodiments, articles are delivered using a device described herein, a canula, syringe, autojector, microneedle patches, self-injection devices and the like. In some embodiments, devices are delivered surgically via incision (e.g., arthroscopic incision).

[0101] In some embodiments, the delivery device further performs joint aspiration or lavage (e.g., during the same procedure as the delivery of the article) and / or delivery of additional active agents (e.g., steroids) or inert agents (e.g., saline, lubricating agents, etc.). The methods described herein can be utilized in conjunction with any additional treatments relevant to the condition or disease.

[0102] While the present disclosure has been illustrated with the treatment of OA in a joint, the compositions and methods described herein find use in treatment of any number of conditions (e.g., related to protection, repair and renewal of damaged tissues). The articles described herein can be utilized in treatment of any number of hard and / or soft tissues. For example, in some embodiments, a decellularized amnion article is delivered or applied to a tissue (e.g., muscle, ligament, tendon, cartilage), bone, wound, eye, nerve, vasculature, or tooth of a subject.

[0103] In some embodiments, the subject is a human subject. In other embodiments, the subject is mammal such as, for example, a cat, dog, cattle, or pig.Examples

[0001] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.Example 1Decellularization of human amnion membrane

[0104] This example describes the development of decellularization methods for treating amnion tissue. Decellularization is a procedure that removes cellular components from a tissue or organ, leaving behind an extracellular matrix scaffold.Methods

[0105] Five protocols for the decellularization of amnion samples obtained from placental tissue were evaluated:

[0106] Protocol number 1 : Freeze thaw cycle + Trypsinization

[0107] Protocol number 2: Freeze thaw cycle +NaOH (with mechanical scrapping using gauze)

[0108] Protocol number 3:NaOH (with mechanical scrapping using gauze)

[0109] Protocol number 4:Trypsin + NaOH (with mechanical scrapping using gauze)

[0110] Protocol number 5: Freeze thaw cycle + Trypsinization + NaOH

[0111] Samples from each protocol were tested for total DNA and collagen quantification as described below.

[0112] The total DNA content in the placental membrane samples was isolated and quantified employing the Quant-iT PicoGreen dsDNA assay kit (Thermofisher Scientific), adhering to the provided protocol by the manufacturer. In summary, following digestion with papain, DNA samples were combined with the Quant-iT PicoGreen reagent. Subsequently, these samples were subjected to excitation at 480 nm, and fluorescence emission intensity was recorded at 520 nm using a spectrofluorometer. Quantification of DNA content was performed based on a standard curve and the results were adjusted for the dry weight of the placental membrane (AM) tissue, calculated as calculated from known concentration of the standard curve / initial tissue weight.

[0113] For DNA quantification assays N= biological replicates (patient samples) were used for analysis. For each biological replicate, N=2 samples of placental tissue were utilized to evaluate total DNA content. For fluorometric analysis, 6 technical repeats were used for samples and 4 technical repeats were used for standard curve.

[0114] The collagen content in lyophilized decellularized placental tissue samples was quantitatively assessed using a fluorometric approach, specifically through a hydroxyproline assay kit provided by Sigma, alongside a hydroxyproline standard. Subsequently, the measured collagen content was normalized to the dry weight of the tissue for standardized comparisons.

[0115] For collagen quantification assays N=3 biological replicates (patient samples) were used for analysis. For each biological replicate, N=2 samples of placental tissue were utilized. For subsequent fluorometric analysis, 6 technical repeats were used for samples and 4 technical repeats were used for standard curve.Results

[0116] FIG. 2 shows data for collagen quantification of decellularized and control amnion patches. This figure illustrates the quantification of collagen normalized to 1 mg of tissue weight among various experimental groups: (1) Control, (2) Freeze-Thaw (FT) + Trypsin, (3) FT + Sodium Hydroxide (NaOH), (4) NaOH alone, (5) Trypsin + NaOH, and (6) FT + Trypsin + NaOH. These groups were evaluated in relation to native amniotic membrane (AM) tissue, designated as the baseline control. For each experimental condition, six replicates (n=6) were analyzed. Statistical analysis was conducted using one-way ANOVA, yielding a P-value of 0.4272. This result indicates a lack of statistically significant differences in collagen content among the experimental groups when compared to each other.

[0117] FIG. 3 shows comparative quantification of total DNA in decellularized treatment groups versus control. This figure presents the quantitative analysis of DNA content among six distinct experimental amnion groups: (1) Control, (2) Freeze-Thaw (FT) + Trypsin, (3) FT + Sodium Hydroxide (NaOH), (4) NaOH alone, (5) Trypsin + NaOH, and (6) FT + Trypsin + NaOH, in comparison to native amnion membrane (AM) tissue, which served as the baseline control. The data demonstrate a statistically significant diminution in DNA content within all groups subjected to decellularization protocols relative to the control group. For each experimental condition, six replicates (n = 6) were analyzed. Statistical evaluation of disparities in DNA content between the treatment groups and the control utilized the Mann- Whitney U test, where a value of *P < 0.05 was considered indicative of statistical significance.

[0118] FIG. 4 shows propidium iodide staining for DNA in control vs NaOH treatment group. The figure illustrates the assessment of DNA content in amnion membrane samples following decellularization treatment using NaOH and mechanical scraping, detected via propidium iodide(PI) staining. The experiment utilized one biological sample (from a single patient), with two samples each for the NaOH treatment and the control group. The figure showcases representative images from areas within the four amnion tissue samples analyzed for DNA content: A) Sample 1 treated with NaOH, B) Sample 2 treated with NaOH, C) Sample 1 from the control group, and D) Sample 2 from the control group. The dye intensity was visually assessed by five observers, indicating that propidium iodide staining appeared significantly more intense in the control samples compared to those treated with NaOH. NaOH treatment does not affect the degradation characteristics of DHAM in comparison to control.

[0119] Protocol number 3 was selected for further experiments. Details of the protocol are as follows: The human placental tissue membrane sample section is transferred to a cell culture environment. The sample is submerged in a 0.5 M NaOH solution, followed by a gentle mechanical abrasion using a gauze pad for 60 seconds. Post-treatment, the sample is transferred to a 15 ml tube and subjected to repeated rinsing and vortexing with Milli-Q water.

[0120] FIG. 10 show images and a flow chart for methods of generating and obtaining decellularized amnion membrane articles.Example 2Biocompatibility studies

[0121] This example describes in-vitro evaluation using a rat chondrocyte cell line to investigate the biocompatibility of the decellularized human amnion membrane. Amnion membrane patches were generated using the methods described in Example 1. An overview of the experimental design is shown in FIG. 7. 3 groups were plated into 24 well plates: Cells alone (positive control); Cells + AM; Cells + IL-10 (negative control) and then contacted with decellularized amnion membranes. Calcein AM was used to stain live cells because it is converted into a green fluorescent compound by esterases present in live cells. Ethidium homodimer was used to stain for dead cells, as it can penetrate cells with compromised membranes and bind to DNA, fluorescing red.

[0122] Results indicated that the decellularized amnion membrane is biocompatible with rat primary chondrocytes. No changes in visualized cells in the presence of the decellularized amnion membrane was observed at 2 days or 3 days of contact.Example 3In Vivo analysis

[0123] This example describes an in vivo evaluation of decellularized amnion membrane patch (dAMP) in a collagenase-induced rat model of osteoarthritis in-vivo analysis. The experimental protocol (FIG. 8) received approval from the Institutional Animal Care Committee at UConn Health. Sprague-Dawley rats, procured from a certified supplier, underwent a minimum acclimatization period of 24 hours prior to any procedures. To induce an osteoarthritis (OA) phenotype, 500 units of collagenase type II were administered intraarticularly on Day 0 and Day 3. Concurrent with each injection, anesthesia was achieved using 2-4% isoflurane to ensure animal welfare. Subsequently, the animals were stratified into three experimental cohorts: one undergoing placental tissue surgical implantation (FIG. 1), a sham surgery group, and a negative control group that did not receive any surgical intervention post-OA induction. 6 weeks after surgery, animals were sacrificed and evaluated for efficacy of the treatment.

[0124] Upon animal sacrifice, knee joints of rats were harvested, decalcified, and preserved in a formalin solution until histological analysis was performed. Contralateral knees (left knee) were taken as healthy controls. Samples were evaluated using immunohistochemical analysis, safranin-0 (staining cartilage), lubricin antibody staining, and H&E.

[0125] Results of safranin O staining are shown in FIG. 5. The results demonstrate that decellularized amnion patches preserve hyaline cartilage.

[0126] Results of lubricin staining using immunohistochemical analysis with PRG4 are shown in FIGS. 9A-B. The results demonstrate that dAMP implantation may induce upregulation of lubricin expression within the joint via acting as a protective barrier from synovial fluid or by inducing expression of lubricin.

[0127] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0128] Clause 1. A decellularized amnion membrane article.

[0129] Clause 2. The article of clause 1, wherein the article is a patch or a sheet.

[0130] Clause 3. The article of clause 1 or 2, wherein the decellularization comprises the use of sodium hydroxide and mechanical scraping.

[0131] Clause 4. The article of clause 3, wherein the mechanical scraping is with surgical gauze.

[0132] Clause 5. The article of any one of the previous clauses, wherein the article has a DNA level below about 50 ng / mg of dry tissue.

[0133] Clause 6. The article of any one of the previous clauses, wherein the amnion membrane is isolated from a subject.

[0134] Clause 7. The article of clause 6, wherein the subject is a human or non-human mammal.

[0135] Clause 8. The article of clause 6 or 7, wherein the amnion membrane is isolated from a placenta.

[0136] Clause 9. The article of any one of the previous clauses, wherein the article is lyophilized.

[0137] Clause 10. A composition, kit, or system comprising the article of any one of clause 1 to 9.

[0138] Clause 11. The composition, kit, or system of clause 10, wherein the composition is a pharmaceutical composition.

[0139] Clause 12. The composition, kit, or system of clause 10 or 11, wherein the article is twisted around an absorbable material.

[0140] Clause 13. The composition, kit, or system of clause 12, wherein the absorbable material is a yam.

[0141] Clause 14. The composition, kit, or system of any one of clauses 10 to 13, wherein the composition, kit, or system fiirther comprises a delivery device.

[0142] Clause 15. The composition, kit, or system of clause 14, wherein the delivery device comprises one or more of a plurality of syringe ports, a plurality of stoppers, a delivery needle, and a plunger.

[0143] Clause 16. The composition, kit, or system of clause 15, wherein the delivery needle is configured to deliver an article to a specific location in a subject.

[0144] Clause 17. The composition, kit, or system of clause 16, wherein the article is preloaded into the delivery device.

[0145] Clause 18. The composition, kit, or system of any one of clauses 10 to 17, wherein the composition further comprises an active agent.

[0146] Clause 19. The composition, kit, or system of clause 18, wherein the active agent is absorbed into the article.

[0147] Clause 20. A delivery device comprising the article of any one of clauses 1 to 9.

[0148] Clause 21. The delivery device of clause 20, wherein the delivery device comprises a plurality of syringe ports, a plurality of stoppers, a delivery needle, and a plunger.

[0149] Clause 22. The delivery device of clause 21, wherein the delivery needle is configured to deliver the article to a specific location in a subject.

[0150] Clause 23. The delivery device of any one of clauses 20 to 22, wherein the delivery device birther comprises or contains one or more of a buffer, saline, a pharmaceutical agent, and / or a bioactive fluid.

[0151] Clause 24. An amnion structure, comprising: A plurality of decellularized amnion membrane articles, wherein each article comprises a plurality of edges, and wherein at least two of the edges of the articles are sealed to each other.

[0152] Clause 25. The structure of clause 24, wherein at least one of the edges of the articles are not sealed.

[0153] Clause 26. The structure of clause 24 or 25, wherein the sealed edges form a bag-like structure.

[0154] Clause 27. The structure of any one of clauses 24 to 26, wherein the structure is filled with or coated with an active agent.

[0155] Clause 28. The structure of clause 27, wherein the active agent is selected from, amniotic fluid, synovial fluid, fat, hyaluronic acid, cells, stem cells, small molecule pharmaceuticals, lubricants, placental fragment, decellularized extracellular matrix, degradable fabric, and electrospun mesh.

[0156] Clause 29. The structure of clause 28, wherein the lubricant is selected from glycerol, PEG, a phospholipids, a poloxamers, a fatty acids, a silicone-based lubricant, polyacrylamide, and a liposome-based lubricant.

[0157] Clause 30. A method of generating a decellularized amnion membrane article, comprising:

[0158] a) obtaining an amnion membrane;

[0159] b) contacting the amnion membrane with abase (e.g., sodium hydroxide); and

[0160] c) mechanically scraping the membrane.

[0161] Clause 31. A method of treating osteoarthritis, comprising delivering the article of any one of clauses 1 to 9 to a joint of a subject in need thereof.

[0162] Clause 32. The method of clause 31, wherein the joint is a knee joint, a wrist, an elbow joint, a spinal vertebrae or disc space, a shoulder joint, hip joint, a toe joint, or a finger joint.

[0163] Clause 33. The method of clause 32, wherein the joint is a knee joint.

[0164] Clause 34. The method of any one of clauses 31 to 33, wherein the article is delivered to the joint via injection (e.g., using a delivery device), surgical implantation, or fixation.

[0165] Clause 35. The method of clause 34, wherein the delivery device further performs joint aspiration.

[0166] Clause 36. The method of any one of clauses 31 to 35, wherein the subject is a human subject.

[0167] Clause 37. The method of any one of clauses 31 to 36, wherein the article preserves or regenerates articular cartilage in the joint.

[0168] Clause 38. The method of any one of clauses 31 to 37, wherein the article induces upregulation of lubricin in the joint.

[0169] Clause 39. A method, comprising delivering the article of any one of clauses 1 to 9 to a subject in need thereof.

[0170] Clause 40. The method of clause 39, wherein the article is delivered to a joint, a tissue, a bone, a wound, an eye, or a tooth of the subject.

[0171] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above - described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.Other Embodiments

[0172] Embodiments disclosed here are not limiting of the subject matter and is merely exemplary. Various other components may be included and called upon for providing for aspectsof the teachings herein. For example, additional materials, combinations of materials and / or omission of materials may be used to provide for added embodiments that are within the scope of the teachings herein.

[0173] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the pre-sent embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A decellularized amnion membrane article.

2. The article of claim 1 , wherein the article is a patch or a sheet.

3. The article of claim 1 or 2, wherein the decellularization comprises the use of sodium hydroxide and mechanical scraping.

4. The article of claim 3, wherein the mechanical scraping is with surgical gauze.

5. The article of any one of the previous claims, wherein the article has a DNA level below50 ng / mg of dry tissue.

6. The article of any one of the previous claims, wherein the amnion membrane is isolated from a subject.

7. The article of claim 6, wherein the subject is a human or non-human mammal.

8. The article of claim 6 or 7, wherein the amnion membrane is isolated from a placenta.

9. The article of any one of the previous claims, wherein the article is lyophilized.

10. A composition, kit, or system comprising the article of any one of claims 1 to 9.

11. The composition, kit, or system of claim 10, wherein the composition is a pharmaceutical composition.

12. The composition, kit, or system of claim 10 or 11, wherein the article is twisted around an absorbable material.

13. The composition, kit, or system of claim 12, wherein the absorbable material is a yam.

14. The composition, kit, or system of any one of claims 10 to 13, wherein the composition, kit, or system fiirther comprises a delivery device.

15. The composition, kit, or system of claim 14, wherein the delivery device comprises a plurality of syringe ports, a plurality of stoppers, a delivery needle, and a plunger.

16. The composition, kit, or system of claim 15, wherein the delivery needle is configured to deliver an article to a specific location in a subject.

17. The composition, kit, or system of claim 16, wherein the article is preloaded into the delivery device.

18. The composition, kit, or system of any one of claims 10 to 17, wherein the composition fiirther comprises an active agent.

19. The composition, kit, or system of claim 18, wherein the active agent is absorbed into the article.

20. A delivery device comprising the article of any one of claims 1 to 19.

21. The delivery device of claim 20, wherein the delivery device comprises a plurality of syringe ports, a plurality of stoppers, a delivery needle, and a plunger.

22. The delivery device of claim 21 , wherein the delivery needle is configured to deliver the article to a specific location in a subject.

23. The delivery device of any one of claims 20 to 22, wherein the delivery device further comprises or contains one or more of a buffer, saline, a pharmaceutical agent, and / or a bioactive fluid.

24. An amnion structure, comprising:A plurality of decellularized amnion membrane articles, wherein each article comprises a plurality of edges, and wherein at least two of the edges of the articles are sealed to each other.

25. The structure of claim 24, wherein at least one of the edges of the articles are not sealed.

26. The structure of claim 24 or 25, wherein the sealed edges form a bag-like structure.

27. The structure of any one of claims 24 to 26, wherein the structure is filled with or coated with an active agent.

28. The structure of claim 27, wherein the active agent is selected from, amniotic fluid, synovial fluid, fat, hyaluronic acid, cells, stem cells, small molecule pharmaceuticals, lubricants, placental fragment, decellularized extracellular matrix, degradable fabric, and electrospun mesh.

29. The structure of claim 28, wherein the lubricant is selected from glycerol, PEG, a phospholipids, a poloxamers, a fatty acids, a silicone-based lubricant, polyacrylamide, and a liposome-based lubricant.

30. A method of generating a decellularized amnion membrane article, comprising a) obtaining an amnion membrane; b) contacting the amnion membrane with a base; and c) mechanically scraping the membrane.

31. The method of claim 30, wherein the base is sodium hydroxide.

32. A method of treating osteoarthritis, comprisingdelivering the article of any one of claims 1 to 9 to a joint of a subject in need thereof.

33. The method of claim 32, wherein the joint is a shoulder joint, a wrist, an elbow joint, a spinal vertebrae or disc space, a knee joint, a hip joint, a toe joint, or a finger joint.

34. The method of claim 33, wherein the joint is a knee joint.

35. The method of any one of claims 32 to 34, wherein the article is delivered to the joint via injection using a delivery device.

36. The method of claim 35, wherein the delivery device further performs joint aspiration.

37. The method of any one of claims 32 to 36, wherein the subject is a human subject.

38. The method of any one of claims 32 to 37, wherein the article preserves articular cartilage in the joint.

39. The method of any one of claims 32 to 38, wherein the article induces upregulation of lubricin in the joint.

40. The method of any one of claims 32 to 39, wherein the delivery is by injection, surgical implantation, or fixation.

41. A method, comprising delivering the article of any one of claims 1 to 9 to a subject in need thereof.

42. The method of claim 41 , wherein the article is delivered to a joint, a tissue, a bone, a wound, an eye, or a tooth of the subject.

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