Method for preparing a final sterile hydrogel or colloidal suspension derived from extracellular matrix and its use.

Abstract

A method for preparing a final sterile hydrogel or colloidal suspension derived from extracellular matrix is ​​provided, along with its use. [Solution] Methods for treating fistulas in a subject, such as, but not limited to, anal fistulas, are disclosed. In some embodiments, the method involves topically administering an effective amount of a composition comprising a mammalian acoustic extracellular matrix (ECM) hydrogel and, optionally, trehalose, to the fistula in the subject. Compositions for use in these methods are also disclosed. In further embodiments, compositions comprising a mammalian acoustic extracellular matrix (ECM) hydrogel for use in treating fistulas in a subject are disclosed.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits under U.S. Provisional Application No. 63 / 077,084, filed September 11, 2020, which is incorporated herein by reference.

[0002] Government support description This invention was made with government support under grant number W81XWH-19-9-0012, awarded by the U.S. Army Medical Research and Materiel Command (ARMY / MRMC). The Government has specific rights to this invention.

[0003] Technical field This application relates to the field of hydrogels, and more particularly, but not limited to, the use of mammalian acoustic extracellular matrix (ECM) hydrogels for repairing fistulas such as anal fistulas.

[0004] Parties to the joint research agreement This invention was made under a research agreement between the University of Pittsburgh Commonwealth System of Higher Education and ECM Therapeutics, Inc., which was carried out prior to the filing date of this application. [Background technology]

[0005] background Anorectal fistulas are pathological connections between the epithelial surface of the anal canal and the skin of the perineal and perianal regions. Anorectal fistulas pose a significant procedural burden for gastroenterology and colorectal surgery due to the differing approaches to their management. Current methods often result in poor procedural outcomes. Regardless of management, anorectal fistulas significantly impact patient morbidity and mortality as fistula complications, ranging from social distress to obvious infections and sepsis. Furthermore, high recurrence rates, surgical complication rates, and subsequent management of anorectal fistulas significantly reduce patients' quality of life and increase healthcare costs. Therefore, new compositions and methods are needed to treat these fistulas and other fistulas. [Overview of the project] [Means for solving the problem]

[0006] Summary of Disclosure A method for treating a fistula in a subject is disclosed. In some embodiments, the method comprises topically administering an effective amount of a composition containing a mammalian acoustic extracellular matrix (ECM) hydrogel to the fistula in the subject, wherein a) the mammalian acoustic ECM hydrogel is thermoreversible, being in the gel phase at temperatures below about 37°C and transitioning to the liquid phase at temperatures above about 37°C, b) the mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of about 0.1 mg / ml to about 1,000 mg / ml, and c) the composition has a storage modulus (G') to loss modulus (G") ratio in the range of about 6:1 to about 12:1 at 37°C. In some non-limiting embodiments, the fistula is an anal fistula.

[0007] In some embodiments, a composition is disclosed comprising i) a mammalian acoustic extracellular matrix (ECM) hydrogel, where a) the mammalian acoustic ECM hydrogel is thermoreversible, being in the gel phase at temperatures below about 37°C and transitioning to the liquid phase at temperatures above about 37°C, b) the mammalian acoustic ECM hydrogel contains solubilized ECM at concentrations of about 0.1 mg / ml to about 1,000 mg / ml, and c) the composition has a storage modulus (G') to loss modulus (G") ratio of about 6:1 to about 12:1 at 37°C. The composition also comprises ii) 0.1 mg / ml to about 700 mg / ml of trehalose, and iii) about 1 to about 30% (by weight per volume) of pulverized ECM that has not been solubilized in the hydrogel. In some non-limiting embodiments, the composition is used in a method for treating a fistula of interest. In some non-limiting embodiments, the fistula is an anal fistula.

[0008] In further embodiments, compositions comprising a mammalian acoustic extracellular matrix (ECM) hydrogel for use in the treatment of a fistula of interest are disclosed. In these compositions, a) the mammalian acoustic ECM hydrogel is thermoreversible, and the mammalian acoustic ECM hydrogel is in the gel phase at temperatures below about 37°C and transitions to the liquid phase at temperatures above about 37°C; b) the mammalian acoustic ECM hydrogel contains solubilized ECM at concentrations of about 0.1 mg / ml to about 1,000 mg / ml; and c) the composition has a storage modulus (G') to loss modulus (G") ratio of about 6:1 to about 12:1 at 37°C. In some non-limiting examples, the fistula is an anal fistula.

[0009] The aforementioned and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which will proceed with reference to the accompanying drawings. Brief explanation of the drawing [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1: Trehalose prevents acoustic ECM hydrogel aggregation after E-beam sterilization. 100 mg / ml dermal ECM (dECM) hydrogels containing 20 or 40 mg / ml of trehalose were prepared using ultrasonic cavitation. 100 mg / ml dermal ECM hydrogels prepared without trehalose were used as a control (left panel). The data show that the addition of trehalose at concentrations of 20–40 mg / ml (center and left panels) prevented the formation of aggregates in the dermal ECM hydrogels after sterilization with a 35 kGy E-beam. Compositions without trehalose were not adhesive and formed small aggregates or clumps, while compositions containing trehalose generally formed a uniform adhesive composition with a smooth texture.

[0011] [Figure 2] Figure 2: Macroscopic gelation test. 5% colloidal dECM hydrogels containing 40 or 66 mg / ml trehalose were subjected to E-beam sterilization at specified doses. After sterilization, the samples were cooled to 4°C and injected into ring molds. Macroscopic evaluation showed that, when injected at low temperatures, the 5% colloidal dECM hydrogel containing 40 mg / ml trehalose formed more aggregates than the 5% colloidal dECM hydrogel containing 60 mg / ml trehalose at all E-beam doses tested.

[0012] [Figure 3] Figure 3: Macroscopic gelation test at 37°C. 5% colloidal hydrogels containing 40 or 66 mg / ml trehalose were subjected to E-beam sterilization at the specified doses. After sterilization, the samples were cooled to 4°C and injected into ring molds. The samples were then placed in a 37°C incubator for 1 hour. Macroscopic evaluation showed that dECM colloidal hydrogels containing trehalose as a radioprotective agent and subjected to E-beam sterilization maintained their shape at 37°C at both trehalose concentrations.

[0013] [Figure 4]Figure 4: Macroscopic gelation test at 37°C with gentle handling. 5% colloidal hydrogels containing 40 or 66 mg / ml trehalose were subjected to E-beam sterilization at the specified dosage. After sterilization, the samples were cooled to 4°C and injected into ring molds. The samples were then placed in a 37°C incubator for 1 hour. After 1 hour, the gels were manipulated by pressing down on the center. Macroscopic evaluation showed that the dECM colloidal hydrogel containing 40 mg / ml trehalose decomposed more easily under gentle handling than the colloidal hydrogel containing 66 mg / ml trehalose.

[0014] [Figure 5A-5B] Figures 5A-5B: Evaluation of viscoelastic properties of skin ECM colloidal hydrogels containing trehalose. (A) 2, 5, or 10% colloidal hydrogels containing 20 or 40 mg / ml trehalose were subjected to E-beam sterilization using a dose of 25 kGy. The graph shows the average storage modulus of the samples. The results show that the sample containing 20 mg / ml trehalose was stiffer than the sample containing 40 mg / ml trehalose, indicating that increasing colloid concentration increases the stiffness of the material. (B) Average storage modulus of 5% colloidal hydrogels containing 66 mg / ml trehalose and sterilized by E-beam at five different doses.

[0015] [Figure 6] Figure 6: Trehalose does not induce the expression of iNOS or Resistin-like β ("RETNLB" or "Fizz1") in macrophages. Mouse bone marrow-derived macrophages were treated with untreated (control) or escalating concentrations of trehalose (8.5–68 mg / ml) for 24 hours, fixed, immunolabeled with strong indicators of pro-inflammatory M1-like macrophage markers (iNos) or remodeling-inducing M2-like macrophage markers (Fizz1), and counterstained with DAPI. Treatment of cells with IFNγ and lipopolysaccharide (LPS) was used as a positive control for the M1-like phenotype, and IL-4 was used as a positive control for the M2-like phenotype. F4 / 80 staining was used as a positive control for macrophages. Cells were imaged at 200x magnification.

[0016] [Figure 7-1] Figures 7A - 7E: A perianal fistula can be filled using the dermal acoustic hydrogel. A transphincteric fistula with a length of 4 cm was created in a pig. The 100 mg / ml dermal acoustic gel was administered by inserting a catheter through the external opening of the conduit (A) and directed towards the internal opening (B). Then, the catheter was slowly withdrawn while injecting the gel into the conduit (C, D). (E) Photograph of the fistula filled with the dermal acoustic gel. The gel is the dark green spot approximately at 11 o'clock above the anus. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

Mode for Carrying Out the Invention

[0017] Detailed Description of Some Embodiments An anorectal fistula is a pathological connection between the epithelial surface of the anal canal and the skin of the perineum and perianal region. There is substantial debate in the approach for managing anorectal fistulas, and due to the poor treatment outcome, it poses a significant treatment burden in gastroenterology and colorectal surgery. Despite management, anorectal fistulas can have a significant impact on the morbidity and mortality of patients as secondary complications of the fistula, and can extend to obvious infectious diseases and sepsis from social confusion. Furthermore, the high recurrence rate, surgical complication rate, and subsequent treatments for managing anorectal fistulas significantly reduce the quality of life of patients and increase medical costs. A space - filling, remodeling - promoting colloidal hydrogel composition that can be used in the treatment of fistulas is disclosed.

[0018] This specification discloses that compositions comprising mammalian acoustic ECM hydrogel have demonstrated clinical efficacy for the management of unregulated inflammation and for promoting wound healing in anal fistulas. As disclosed in the examples, ECM was exposed to sonication and used to produce compositions for use in treating anal fistulas. In vitro results demonstrate that the disclosed compositions, when sterilized, can fill fistula tracts of complex structure at body temperature, can regulate macrophages toward a remodeling-promoting phenotype (Fizz+), and can be used as a rigid biomaterial with hemostatic properties. The disclosed compositions maintained rigidity in ex vivo tracts, did not decompose at body temperature, and did not leak from the tracts.

[0019] In a postmortem in vitro pig fistula model, the compositions were able to fill the ducts at body temperature without leakage. The combination of space-filling, anti-inflammatory, and hemostatic properties allowed these compositions to be used for the treatment of simple and complex anorectal fistulas. Ease of injection, sterility, and stability make the disclosed compositions available for "off-the-shelf" use in both surgical and outpatient settings. The disclosed compositions improve healing, mitigate surgical bleeding, reduce complications, and decrease recurrence in the management of anorectal fistulas. term

[0020] Unless otherwise specified, technical terms will be used according to their conventional usage. Definitions of common terms in molecular biology can be found in Krebs et al (Eds.), Lewin's Genes XII, published by Jones & Bartlett Publishers, 2017; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008, and other similar references. To facilitate the consideration of various embodiments of this disclosure, the following explanations of specific terms are provided.

[0021] Acid proteases are enzymes that cleave peptide bonds, and whose activity in cleaving peptide bonds increases at acidic pH. For example, but are not limited to, acid proteases may include pepsin and trypsin.

[0022] Anal fistula: A small tunnel connecting the anal canal to the perianal skin. Most are associated with anorectal abscesses. Anal fistulas can result from an infection of the anal glands (anal glad) that spreads to the skin. Symptoms include pain, swelling, and discharge of blood or pus from the anus. There are several types of anal fistulas, including intersphincteric (most common), penetrating, extrasphincteric, and suprasphincteric (least common).

[0023] Antibiotics: Compounds or substances that kill or substantially slow the growth of bacteria, fungi, or any other microorganisms. "Antibacterial" means that a compound or substance kills or substantially slows the growth of bacteria.

[0024] Antimicrobial antibiotics are generally classified based on their mechanism of action, chemical structure, or activity spectrum. Most target bacterial function or growth processes. Those that target bacterial cell walls (e.g., penicillins and cephalosporins) or cell membranes (e.g., polymyxins), or those that interfere with essential bacterial enzymes (e.g., quinolones and sulfonamides) are bactericidal. Those that target protein synthesis (e.g., aminoglycosides, macrolides, and tetracyclines) are generally bacteriostatic. Further classification is based on their target specificity.

[0025] "Narrow-spectrum" antimicrobial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria. "Broad-spectrum" antibiotics affect many different types of bacteria. Antimicrobial agents also include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), and oxazolidinones (such as linezolid).

[0026] Topical antibiotics are antibiotics applied to the body surface, such as the skin or eyes. Topical antibiotics are often formulated as ointments or creams and contain active agents such as macrolide antibiotics (erythromycin, etc.), sulfonamide antibiotics (sulfacetamide, etc.), cyclic peptides (bacitracin, polymyxin, etc.), pseudodomonic acid (mupirocin, etc.), aminoglycosides (neomycin, etc.), or quinolones (ciprofloxacin or ofloxacin, etc.), nitroimidazoles (metronidazole, etc.), or combinations of drugs (bacitracin / polymyxin or neomycin / polymyxin B / bacitracin, etc.).

[0027] Biocompatibility: Any material that, when implanted in a mammalian subject, does not cause an adverse reaction in the subject. A biocompatible material, when introduced into an individual, can perform its intended function, is not toxic or harmful to that individual, and does not induce immunological rejection of the material in the subject.

[0028] Centrifugal separation is the process by which centrifugal force is applied to a mixture, thereby causing the denser components of the mixture to move away from the axis of the centrifuge compared to the other less dense components in the mixture. The force applied to the mixture is a function of the velocity of the centrifuge rotor and the radius of spin. In most applications, the force of the spin causes the precipitate (pellet) to collect at the bottom of the centrifuge tube, and the remaining solution is called the "supernatant" or "clear liquid," as appropriate. In other similar applications, density-based separation or "gradient centrifugation" techniques are used to isolate specific species from mixtures that contain both denser and less dense components than the desired component.

[0029] During the circular motion of a centrifuge rotor, the force acting on it is the product of the spin radius and angular velocity, and this force is traditionally expressed as acceleration relative to the standard acceleration "g" due to gravity on the Earth's surface. The centrifugal force acting on it is called the "relative centrifugal force" (RCF) and is expressed as a multiple of "g".

[0030] Grinding (grinding and grinding): A process that reduces larger particles to smaller particles, including but not limited to grinding, blending, shredding, slicing, milling, or cutting. ECM can be ground in any form, including but not limited to hydrated, frozen, air-dried, freeze-dried, powder, or sheet form. "Ground ECM" contains intact collagen. Ground ECM is not exposed to ultrasound.

[0031] Contact: Arrangement in a direct physical meeting, which may be in solid or liquid form.

[0032] Cytokines: The term "cytokines" is used as a general term for a diverse group of soluble proteins and peptides that act as fluid regulators at nano- to picomolar concentrations, modulating the functional activity of individual cells and tissues under normal or pathological conditions. These proteins also directly mediate intercellular interactions and regulate processes occurring in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor-α, interleukin (IL)-6, IL-10, IL-12, transforming growth factor, and interferon-γ.

[0033] Diagnosis: The process of identifying a disease based on its signs, symptoms, and the results of various tests. The conclusion reached through this process is also called a "diagnosis." Common types of tests include blood tests, medical imaging, and biopsies.

[0034] Extracellular matrix (ECM): A natural cell-free scaffold for cell growth. Natural ECM (ECM found in mammals and multicellular organisms such as humans) is a complex mixture of structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines, and growth factors. In mammals, ECM often contains approximately 90% collagen in its various forms. The composition and structure of ECM vary depending on the tissue source. For example, the submucosa of the small intestine (SIS), bladder matrix (UBM), esophagus (E), and liver stromal ECM each differ in their overall structure and composition due to the unique cellular niches required by each tissue. The intact "extracellular matrix" and "intact ECM" are extracellular matrices that retain the activity of their structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial agents, chemoattractants, cytokines, and growth factors.

[0035] The structure and / or activity of biomolecules within the ECM can be altered or removed chemically or mechanically, for example, by crosslinking and / or dialysis of the ECM. An intact ECM is essentially unenzymatically digested, uncrosslinked and / or dialysis, meaning the ECM has not been subjected to conditions other than digestion, dialysis and / or crosslinking processes, or processes that occur naturally during the storage and handling of the ECM before solubilization. Therefore, a substantially crosslinked and / or dialysis-treated ECM (by means other than commonplace methods that do not substantially affect the gelation and functional properties of the ECM in its use as described herein) is not considered “intact.” “Cell-free” refers to an ECM produced from source tissue that has been treated to remove cells so that the ECM remains. Decellularized tissue is used to produce ECM hydrogels.

[0036] A fistula is an abnormal connection or passage between organs or blood vessels that are not normally connected. Fistulas can occur in various parts of the body, in addition to congenital malformations, deformities, and chromosomal abnormalities, and are associated with diseases of the circulatory, respiratory, digestive, reproductive, musculoskeletal, and connective tissue systems. Fistulas can be classified as blind if they have only one opening, complete if they have both external and internal openings, or incomplete if they have an external skin opening that does not connect to any internal organs. The most common form of fistula is a tube that may have multiple branches.

[0037] A gel is a state of matter between liquid and solid, generally defined as a cross-linked polymer network that swells in a liquid medium. Typically, a gel is a two-phase colloidal dispersion containing both solid and liquid, with a greater amount of solid than in a two-phase colloidal dispersion called a "sol." Thus, a "gel" possesses some of the properties of a liquid (i.e., its shape is elastic and deformable) and some of the properties of a solid (e.g., its shape is sufficiently separated to maintain three dimensions on a two-dimensional surface). "Gelization time," also called "gel time," refers to the time it takes for a composition to become non-flowable under moderate stress.

[0038] Gelation: Formation of a gel from a sol.

[0039] Hemostasis: Suppression or cessation of bleeding.

[0040] Hydrogels: A network of hydrophilic polymer chains, sometimes found as colloidal gels with water as the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also possess a degree of flexibility similar to that of natural tissues. "Acoustic" hydrogels, such as acoustic ECM hydrogels, are produced using ultrasonic energy. The characteristics of these hydrogels are disclosed herein. For hydrogels, the storage modulus (G') is typically about an order of magnitude greater than the loss modulus (G''). "Enzymatic" ECM hydrogels are produced from enzymatically digested ECM. The viscosity of enzyme hydrogels increases when heated to a physiological temperature close to approximately 37°C. For example, enzyme hydrogels are formed from solutions that are injectable at temperatures below 37°C, forming a gel at a physiological temperature of 37°C.

[0041] Isolated: “Isolated” biological components (such as extracellular matrix) are substantially separated from, produced separately from, or purified from other biological components, cells or organisms in which the components naturally occur, i.e., living cells, other chromosomes and extrachromosomal DNA and RNA, and proteins. Therefore, “isolated” ECM includes ECM removed from tissue by standard purification methods. Isolated ECM is isolated from cells that produce ECM.

[0042] Isotonic buffer solution: A solution buffered to a pH between 7.2 and 7.8 and containing a salt at equilibrium concentration to promote an isotonic environment.

[0043] Macrophages are a type of white blood cell that phagocytoses and breaks down cellular debris, foreign substances, microorganisms, and cancer cells. In addition to their role in phagocytosis, these cells play a crucial role in development, tissue maintenance, and repair, as well as in both innate and adaptive immunity, by mobilizing and influencing other cells, including immune cells such as lymphocytes. Macrophages can exist in many phenotypes, including those called M1 and M2 (also known as "M1-like" and "M2-like" phenotypes). Macrophages that primarily perform pro-inflammatory functions are called M1 macrophages (CD86+ / CD68+), while macrophages that reduce inflammation and promote and regulate tissue repair are called M2 macrophages (CD206+ / CD68+). Markers for identifying the various phenotypes of macrophages differ by species. Note that macrophage phenotypes are represented by a spectrum ranging between the extreme values ​​of M1 and M2. The marker Fizz-1 (see Raes et al., Dev. Immunol. 9:151-159, 2002, incorporated herein by reference) identifies macrophages that are thought to be undergoing remodeling, i.e., M2 macrophages.

[0044] Mammals: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human subjects and veterinary subjects.

[0045] Prevention or treatment of disease: “Preventing” a disease means, for example, inhibiting the partial or complete onset of the disease in a person known to be predisposed to the disease. “Treatment” means a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. In some embodiments, treatment means a reduction in incontinence, closure of at least a portion of a fistula, or a reduction in the symptoms of an anal fistula.

[0046] Solubilized ECM: ECM treated with ultrasonic cavitation, thereby inducing microstructural changes through the physical disruption of protein aggregates.

[0047] Therapeutic agents: Used in a general sense, including procedural agents, prophylactic agents, and replacement agents. "Procedure" or "to treat" means providing a patient with a substance, such as the disclosed composition, in an amount sufficient to measurably affect a biological parameter, such as hemostasis or increased tissue growth.

[0048] Therapeutic Dose: The “therapeutic dose” of a composition means the amount that, when administered to a patient, is effective in providing a therapeutic benefit such as improvement of symptoms, reduction of progression, or regression of the disease. The amount of the composition is sufficient to achieve the desired effect in the subject being treated, for example, a subject with an anal fistula. The therapeutic dose can be administered topically to the anal fistula, etc. Furthermore, the effective dose can be administered as a single dose or in several doses at different times. The effective dose depends on the preparation applied, the subject being treated, the severity and type of the disease, for example, the type of fistula. The compositions used in the methods disclosed herein have equivalent uses in medical and veterinary contexts. Therefore, the general terms “subject” or “patient” are understood to include all animals, including but not limited to humans or veterinary subjects, such as other primates, dogs, cats, horses, and cattle.

[0049] Thermoreversible hydrogels: Hydrogels formed by the entanglement of polymer chains, in which viscosity changes at a temperature characteristic of gelation. The disclosed acoustic ECM hydrogel is a thermoreversible hydrogel that exhibits gelation (transition from sol to gel) upon cooling.

[0050] Topical application: Topical agents are applied only to specific areas and not to the entire body. In certain cases, the composition is applied to the skin or eyes in an area where hemostasis is desired. For example, a pharmaceutical composition may be applied as a topical preparation to wounds, such as epithelial wounds or defects, such as traumatic or surgical wounds, such as abrasions or surgical incisions of the skin or cornea.

[0051] Ultrasonic treatment: A process of exposure to ultrasound with frequencies higher than 20 kHz.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Furthermore, it should be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximations and are provided for illustrative purposes only. “Approximately” indicates within 5% of the listed values. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are listed below. The term “comprises” means “includes.” All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of conflict, this specification, including the definitions of terms, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting. Extracellular matrix (ECM)

[0053] Any type of extracellular matrix can be used to produce mammalian acoustic ECM hydrogels (referring to U.S. Patent Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; (Nos. 5,866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562; 6,890,563; 6,890,564; and 6,893,666). In certain embodiments, the ECM is isolated from vertebrates, mammals including, but not limited to, humans, monkeys, horses, pigs, cattle, and sheep. In certain non-specific examples, ECM is pig.

[0054] ECM may originate from any organ or tissue, including but not limited to the bladder, intestines (small or large intestine, etc.), heart, kidneys, uterus, brain, blood vessels, lungs, bones, muscles, pancreas, stomach, spleen, adipose tissue, muscle tissue, liver, esophagus, placenta, and dermis. ECM can be obtained from cell cultures. In one embodiment, the ECM is isolated from the bladder. In another embodiment, the ECM is derived from the esophagus. In yet another embodiment, the ECM is from the dermis. In yet another embodiment, the ECM is derived from the submucosa (SIS) of the small intestine. The ECM may or may not include a basement membrane portion. In certain embodiments, the ECM includes at least a portion of the basement membrane. The ECM can be produced by decellularizing tissue, for example, removing cells and cellular material from a source tissue or organ. It is desirable to use decellularized material, such as when the ECM is embedded in a target as a component of a hydrogel disclosed herein, in order to prevent an immune response. Removing cellular material, such as when forming hydrogels using ECM, prevents such immune responses.

[0055] U.S. Patent No. 8,361,503 (incorporated herein by reference) discloses the preparation of a bladder extracellular membrane (ECM) such as that of a pig bladder. The ECM is prepared by abrading the bladder tissue using a scalpel handle and a longitudinal wiping motion with moistened gauze to remove the outer layers, including both the serosal and muscular layers. Following the abduction of the tissue segments, the same wiping motion is used to detach the luminal portion of the mucosa from the underlying tissue. In some embodiments, perforation of the submucosa is prevented. After these tissues have been removed, the resulting ECM consists mainly of submucosa.

[0056] The generation of hydrogels from the cutaneous ECM is incorporated herein by reference to Wolf This is disclosed in et al., Biomaterials 33:7028-7038, 2012. The production of ECM from esophageal tissue is described, for example, in Badylak et al., J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005 September;128(1):87-97, 2005, both of which are incorporated herein by reference. U.S. Patent No. 6,893,666, incorporated herein by reference, discloses the production of ECM from the bladder, skin, esophagus and small intestine. ECM may be produced from any of these tissues.

[0057] Commercially available ECM preparations can also be used. In one embodiment, the ECM is derived from the submucosa of the small intestine or SIS. Commercially available preparations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS®, and STRATASIS-ES® (Cook Urological Inc., Indianapolis, Indiana), and GRAFTPATCH® (Organogenesis Inc., Canton, Massachusetts). In another embodiment, the ECM is derived from the dermis. Commercially available preparations include, but are not limited to, PELVICOL® (sold as PERMACOL® in Europe; Bard, Convington, Georgia), REPLIFORM® (Microvasive; Boston, Massachusetts), and ALLODERM® (LifeCell; Branchburg, New Jersey). In yet another embodiment, the ECM is derived from the bladder. Commercially available preparations include, but are not limited to, UBM (Acell Corporation; Jessup, Maryland).

[0058] Tissue for the preparation of ECM can be harvested in a wide variety of ways, and once harvested, various parts of the harvested tissue can be used. ECM can also be prepared from the esophagus and small intestine, as described, for example, in Keane et al., Tissue Eng. Part A, 21(17-18):2293-2300, 2015, which is incorporated herein by reference. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscular layer, digesting the mucosa with a trypsin-containing buffer, and subsequently exposing it to sucrose, TRITON®-X100®, deoxycholic acid, peracetic acid, and DNAse. Small intestinal submucosa (SIS) can be prepared by mechanically removing the mucosa, serosal layer, and superficial layer of the muscular layer from an intact small intestine, leaving the submucosa, muscular mucosa, and compact basal layer intact. The SIS is then treated with peracetic acid. An exemplary protocol is provided in Keane et al. Skin hydrogels can be manufactured, for example, as disclosed in Wolf et al, J Biomed Mater Res A.2013.35(25):6838-49.PMID:23873846.PMCID:3808505, which is incorporated herein by reference.

[0059] In one embodiment, the extracellular matrix (ECM) is isolated from a collected pig bladder to prepare the bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. The serosa, muscularis exostata, submucosa, and most muscularis mucosa can be removed by mechanical abrasion or by a combination of enzymatic treatment, hydration, and abrasion. Mechanical removal of these tissues can be achieved by abrasion using longitudinal wiping motions to remove the outer layers (particularly the anti-luminal smooth muscle layer) and even the luminal portion of the mucosa (epithelial layer). Mechanical removal of these tissues can be achieved, for example, by removing the mesenteric tissue using Adson-Brown forceps and Metzenbaum scissors, and wiping away the muscularis and submucosa using longitudinal wiping motions with a scalpel handle wrapped in moistened gauze or other rigid object. Epithelial cells of the mucosa can also be dissociated by immersing the tissue in a deepithelializing solution, e.g., hypertonic saline, but not limited to these. The resulting UBM comprises a mucosal basement membrane and adjacent mucosal lamina propria, which are further treated with peracetic acid, freeze-dried, and powdered; see U.S. Patent No. 8,361,503 incorporated herein by reference.

[0060] Dermal sections can be used in the preparation of ECM hydrogels (see PCT application 2015 / 15164728, incorporated herein by reference). In certain non-limiting cases, the dermis can be decellularized using 0.25% trypsin / 1% TRITON(registered trademark)-X(registered trademark)-100 (i.e., without SDS) in the following solutions on a vortex shaker at 300 RPM at room temperature: 0.25% trypsin for 6 hours, once; deionized water, 15 minutes, three times; 70% ethanol for 10-12 hours, once; 3% H2O2 for 15 minutes, once; deionized water for 15 minutes, twice; 1% TRITON(registered trademark)-X(registered trademark)-100 in 0.26% EDTA / 0.69% Tris for 6 hours, once, then overnight, once; deionized water for 15 minutes, three times; 0.1% peracetic acid / 4% ethanol for 2 hours, once; PBS for 15 minutes, twice; and finally deionized water for 15 minutes, twice. Next, the dermal sheets were freeze-dried and then processed into fine particles using a Waring blender and a Wiley Mill equipped with a #20 mesh screen.

[0061] In some embodiments, epithelial cells may first be exfoliated by immersing the tissue in a deepithelializing solution, such as hypertonic saline, e.g., 1.0 N saline, for a period ranging from 10 minutes to 4 hours. Exposure to hypertonic saline effectively removes epithelial cells from the underlying basement membrane. The tissue remaining after the initial exfoliation treatment includes the epithelial basement membrane and the tissue layers antiluminal to the epithelial basement membrane. This tissue is then subjected to further treatment to remove most of the antiluminal tissue rather than the epithelial basement membrane. The outer serosa, adventitia, smooth muscle tissue, submucosa, and most muscular mucosa are removed from the remaining deepithelialized tissue by mechanical abrasion or a combination of enzymatic treatment, hydration, and abrasion.

[0062] In some embodiments, the ECM itself can be sterilized by any number of standard techniques, including, but not limited to, exposure to peracetic acid, low-dose gamma irradiation, gas plasma sterilization, ethylene oxide treatment, or electron beam treatment. More typically, sterilization of the ECM is achieved by immersion for 2 hours in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water. The peracetic acid residue is removed by washing twice for 15 minutes with PBS (pH=7.4) and twice for 15 minutes with sterile water. The ECM material can be sterilized by propylene oxide or ethylene oxide treatment, gamma irradiation (0.05–4 mRad), gas plasma sterilization, peracetic acid sterilization, or electron beam treatment. The ECM can also be sterilized by treatment with glutaraldehyde, which causes crosslinking of the protein material, but this treatment substantially alters the material, causing it to be slowly reabsorbed or not reabsorbed at all, inducing a different kind of host remodeling that is more similar to scar tissue formation or encapsulation than structural remodeling. Crosslinking of protein materials can also be induced by carbodiimide, dehydration heat, or photo-oxidation. Disinfect the ECM by immersing it for 2 hours in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol, and 96% (v / v) sterile water, as disclosed in U.S. Patent No. 8,361,503. Then wash the ECM material twice for 15 minutes with PBS (pH=7.4) and twice for 15 minutes with deionized water.

[0063] Generally, after isolation of the target tissue, decellularization is carried out by various methods, such as exposure to hypertonic saline, peracetic acid, TRITON®-X®, or other detergents, though not limited to these methods. Sterilization and decellularization can be performed simultaneously. For example, sterilization with peracetic acid, as described above, can also be used for decellularization, though not limited to these methods. The ECM can then be dried by either freeze-drying or air-drying. The dried ECM can be pulverized by methods including, but not limited to, tearing, milling, cutting, cutting, and shearing. The pulverized ECM can be further processed into a powder form by methods such as cutting or milling in a frozen or freeze-dried state, though not limited to these methods.

[0064] Mammalian ECMs are also commercially available. These include AVITENE®, MICROMATRIX®, and XENMATRIX®. These commercial products can also be used to manufacture mammalian acoustic ECM hydrogels. Acoustic ECM hydrogels and compositions for use

[0065] A composition is disclosed herein that can be administered topically to a fistula, such as an anal fistula, and therefore used in treatment. The composition comprises a mammalian acoustic extracellular matrix (ECM) hydrogel that is thermoreversible, exists in a gel phase at temperatures below about 37°C, and transitions to a liquid phase at temperatures above about 37°C. The mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of about 0.1 mg / ml to about 1,000 mg / ml, and the composition has a storage modulus (G') to loss modulus (G") ratio in the range of about 6:1 to about 12:1 at 37°C. The composition may also contain a radioprotective agent such as trehalose at a concentration of 0.1 mg / ml to 700 mg / ml. The mammalian acoustic ECM hydrogel is disclosed, for example, in PCT Publication No. 2020 / 186082, which is incorporated herein by reference.

[0066] These acoustic ECM hydrogels can be fabricated from any mammalian ECM disclosed above. The source of the ECM may be, for example, a pig, a cattle, a human, or a sheep. In a specific non-limiting example, the ECM is a pig ECM. In another non-limiting example, the ECM is a bladder ECM, a small intestinal submucosal ECM, an esophageal ECM, a tracheal ECM, a liver ECM, or a cutaneous ECM. In one embodiment, the ECM is a bladder ECM. In another embodiment, the ECM is a cutaneous ECM. In yet another embodiment, the ECM is a small intestinal submucosal ECM.

[0067] In some embodiments, the mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration greater than about 0.1 mg / ml. The mammalian acoustic ECM hydrogel can contain solubilized ECM at a concentration of about 0.1 mg / ml to about 1,000 mg / ml. Appropriate concentrations also include solubilized ECM at concentrations of about 1 mg / ml to about 1,000 mg / ml, about 1 mg / ml to about 100 mg / ml, about 10 mg / ml to about 100 mg / ml, about 10 mg / ml to about 200 mg / ml, about 100 mg / ml to about 500 mg / ml, about 50 mg / ml to about 150 mg / ml, about 20 mg / ml to about 70 mg / ml, or about 40 mg / ml to about 66 mg / ml. In one embodiment, the ECM hydrogel can contain solubilized ECM at a concentration of about 20 mg / ml to about 100 mg / ml. Mammalian acoustic ECM hydrogels can contain solubilized ECM in a liquid such as a buffer at concentrations ranging from approximately 10 mg / ml to approximately 500 mg / ml. Mammalian acoustic ECM hydrogels may contain solubilized ECM at concentrations of 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200 mg / ml. Exemplary concentrations include solubilized ECM at concentrations of approximately 20 mg / ml, 40 mg / ml, 66 mg / ml, 70 mg / ml, and 150 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 20 mg / ml to approximately 70 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 40 mg / ml or approximately 66 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 10 mg / ml to approximately 100 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 50 mg / ml to approximately 150 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 10 mg / ml to approximately 200 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at concentrations of approximately 10 mg / ml to approximately 500 mg / ml.

[0068] Exemplary concentrations include solubilized ECM at approximately 20 mg / ml, 40 mg / ml, 66 mg / ml, 70 mg / ml, and 150 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at approximately 20 mg / ml to approximately 70 mg / ml. In a non-limiting example, mammalian acoustic ECM hydrogels contain solubilized ECM at approximately 40 mg / ml to approximately 66 mg / ml.

[0069] In some embodiments, the mammalian acoustic ECM hydrogel contains solubilized ECM at concentrations of about 25 mg / ml to about 600 mg / ml. In further embodiments, the mammalian acoustic ECM hydrogel contains solubilized ECM at concentrations of about 20 mg / ml to about 600 mg / ml, about 25 mg / ml to about 300 mg / ml, about 25 mg / ml to about 200 mg / ml, and about 25 mg / ml to about 150 mg / ml. In further embodiments, the mammalian acoustic ECM hydrogel contains solubilized ECM at concentrations of about 50 mg / ml to about 600 mg / ml. The mammalian acoustic ECM hydrogel may also contain solubilized ECM at concentrations of about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml, about 50 mg / ml to about 150 mg / ml, about 50 to 100 mg / ml, or about 100 to 150 mg / ml. In some non-limiting examples, mammalian acoustic ECM hydrogels are approximately 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, 100-105, 105-110, 110-115, 11 It contains solubilized ECM at concentrations of 5-120, 120-125, 125-130, 130-135, 135-140, 140-145, 145-150, 150-155, 155-160, 160-165, 165-170, 170-175, 175-180, 180-185, 185-190, 190-195, and 195-200 mg / ml.

[0070] In some embodiments, to produce a mammalian acoustic ECM hydrogel, pulverized ECM, such as mammalian ECM, is diluted to a specific concentration in a liquid. The ECM may or may not be freeze-dried before pulverization. The ECM can be pulverized, for example, by grinding, chopping, or cutting. The ground ECM should have pieces ranging from approximately 10 μm to 5000 μm, 10 μm to 4000 μm, 10 μm to 3000 μm, 10 μm to 2000 μm, 10 μm to 1000 μm, 10 μm to 500 μm, 30 μm to 300 μm, 40 to 400 μm, 25 μm to 500 μm, 50 μm to 500 μm, 100 μm to 300 μm, 10 μm to 50 μm, or 10 μm to 100 μm. In one embodiment, the ECM is provided in pieces ranging from approximately 10 μm to 1000 μm. In another embodiment, the ECM is provided in pieces ranging from approximately 10 μm to 2000 μm. In one non-specific example, the fragments range in size from approximately 30 μm to approximately 300 μm.

[0071] The liquid may be a buffer with a neutral pH, for example, about 7.0–7.6, about 7.1–7.5, about 7.2–7.4, about 7.0–7.2, about 7.0–7.4, about 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. The ECM may be diluted with an isotonic buffer such as phosphate-buffered saline (PBS) or Tris-buffered saline, but not limited to these. In some embodiments, the buffer has an osmolality of about 290 mOsm / L. The liquid may be water. In some embodiments, but not limited to these, an isotonic buffer containing phosphate-buffered saline (PBS) may be used to bring the solution to a target pH or to help maintain the pH and ionic strength of the gel to a target level such as physiological pH and ionic conditions. This forms a liquid ECM solution.

[0072] Methods used to prepare mammalian acoustic ECM hydrogels generally do not involve the use of acidic proteases, including pepsin, trypsin, or hyaluronidase, to solubilize the ECM. See PCT application number International Publication 2015 / 164728, incorporated herein by reference. Generally, these methods do not involve contact between the solubilized ECM in liquid and acidic proteases. Therefore, mammalian acoustic ECM hydrogels do not contain exogenous proteases or inactivated exogenous proteases. In some embodiments, mammalian acoustic ECM hydrogels do not contain exogenous pepsin, trypsin, and / or hyaluronidase, or inactivated forms of exogenous pepsin, trypsin, or hyaluronidase.

[0073] ECM in a liquid such as buffered saline is treated with an ultrasonic frequency to solubilize the ECM, and a mammalian acoustic ECM hydrogel containing the solubilized ECM is produced. In one embodiment, the ultrasonic frequency is approximately 20 kHz to approximately 100 kHz. ECM in liquid can be treated with ultrasonic frequencies of approximately 20 kHz to approximately 30 kHz, approximately 20 kHz to approximately 40 kHz, approximately 20 kHz to approximately 50 kHz, approximately 20 kHz to approximately 60 kHz, approximately 20 kHz to approximately 70 kHz, approximately 20 kHz to approximately 80 kHz, or approximately 20 kHz to approximately 90 kHz. ECM in liquid can be treated with ultrasonic frequencies of approximately 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, or 100 kHz. In a non-limiting example, ECM in liquid can be treated with ultrasonic frequencies of approximately 20 kHz.

[0074] The ECM in a liquid such as buffered saline is treated with ultrasound for at least 20 seconds, for example, at least 30 seconds. The ECM in a liquid such as buffered saline is treated with ultrasound for at least 60 seconds. In some embodiments, the ECM in a liquid is treated with ultrasound for at least 60 seconds to about 1 hour. In further embodiments, the ECM in a liquid is treated with ultrasound for at least 60 seconds to about 30 minutes. In further embodiments, the ECM in a liquid is treated with ultrasound for at least 30 seconds to about 30 minutes. In further embodiments, the ECM in a liquid is treated with ultrasound for at least 60 seconds to about 15 minutes. In further embodiments, the ECM in a liquid is treated with ultrasound for at least 30 seconds to about 15 minutes. In some embodiments, the ECM in a liquid is treated with ultrasound for at least 60 seconds to about 10 minutes. In some embodiments, the ECM in a liquid is treated with ultrasound for at least 30 seconds to about 10 minutes. In some embodiments, the ECM in a liquid is treated with ultrasound for at least 60 seconds to about 5 minutes. In some embodiments, the ECM in a liquid is treated with ultrasound for at least 30 seconds to about 5 minutes. ECM in liquid can be treated with ultrasound for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. In some embodiments, ECM in liquid is treated with pulsed ultrasound over the total time enumerated herein. Therefore, in some embodiments, the ECM in a liquid such as buffered saline is treated with pulses of at least about 30 seconds in length, for example, about 30 seconds, about 40 seconds, or about 60 seconds. The ECM in a liquid such as buffered saline can be treated with ultrasound 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, resulting in a total treatment time of 60 seconds to 1 hour, or any of the listed total times.ECM in saline or other liquids can be treated for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 seconds. ECM in saline or other liquids can be treated for at least 30 seconds. Generally, when multiple treatments are used, they are performed over a period of less than one hour. An exemplary method is a 30-second ultrasound pulse followed by 30-45 seconds of no treatment, followed by another treatment. This treatment may be applied 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more. One exemplary, non-limiting method involves six 30-second pulses of ultrasound at approximately 20 kHz, followed by a 45-second off, repeated six times, for a total of three minutes of ultrasound treatment.

[0075] Ultrasound can have an amplitude of approximately 20 μm to approximately 320 μm. Generally, the amplitude is measured from the center of the probe used to generate the ultrasound. The amplitude of the probe's vibrating plane is the distance between the fully extended and fully retracted states of the probe, and is measured in microns (μm). In some embodiments, the amplitude is approximately 30 μm to approximately 200 μm. In further embodiments, the amplitude is approximately 36 μm to approximately 180 μm. The amplitude can be approximately 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 150, 160, 70, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μm. In some embodiments, the amplitude is approximately 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm, 150-160 μm, 160-17 The amplitude can be 0 μm, 170-180 μm, 180-190 μm, 190-200 μm, 200-210 μm, 210-220 μm, 220-230 μm, 230-240 μm, 240-250 μm, 250-260 μm, 260-270 μm, 270-280 μm, 280-290 μm, or 290-300 μm. In a specific, non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm. In a further non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm, and the procedure lasts a total of approximately 1, 2, 3, 4, or 5 minutes, for example, approximately 3 minutes. The ultrasonic treatment may last approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. The ultrasonic treatment may last from approximately 30 seconds to approximately 5 minutes. The ultrasonic treatment may last, for example, from approximately 1 to approximately 5 minutes. The ultrasonic treatment may last from approximately 1 to approximately 10 minutes. The ultrasonic treatment may last, for example, from 1 to approximately 20 minutes. In more embodiments, the ultrasonic treatment may last less than approximately 1 hour, less than approximately 30 minutes, less than approximately 20 minutes, or less than approximately 10 minutes. In some embodiments, the ultrasonic treatment may last at least 30 seconds.In other embodiments, the ultrasonic treatment may range from about 10 minutes to about 24 hours, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the ultrasonic treatment may last up to 48 hours.

[0076] In some embodiments, the ECM in liquid is treated ultrasonically at a temperature in the range of about 30°C to about 43°C. In one embodiment, the ECM in liquid is treated ultrasonically at a temperature in the range of about 35°C to about 40°C. In another embodiment, the ECM in liquid is treated ultrasonically at a temperature in the range of about 36°C to about 38°C. In yet another embodiment, the ECM in liquid is treated ultrasonically at a temperature in the range of about 37°C or higher, for example, about 37°C to about 55°C, for example, about 37°C to about 50°C, for example, about 37°C to about 45°C, for example, about 37°C to about 40°C. The ECM in liquid is treated ultrasonically at a temperature of about 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C. In further embodiments, the ECM in liquid is treated with ultrasound at temperatures above approximately 38°C, for example, approximately 38°C to approximately 50°C, for example, approximately 38°C to approximately 45°C, for example, approximately 38°C to approximately 40°C.

[0077] Ultrasonic treatment produces mammalian acoustic ECM hydrogels containing solubilized ECM. Acoustic ECM hydrogels generally undergo a phase transition from sol to gel at approximately 37°C, and thus transition to a liquid phase above 37°C and to a gel phase below 37°C. At 37°C, mammalian acoustic ECM hydrogels are sufficiently viscous to resemble a gel, but as the temperature rises above 37°C, the gel transitions to a sol. Mammalian acoustic ECM hydrogels form a gel (sol-to-gel transition) when the temperature decreases below 37°C. Therefore, in some embodiments, after sonication, the mammalian acoustic ECM hydrogel is cooled to a temperature below 37°C, for example, about 4°C to about 36°C. Acoustic ECM hydrogels can be cooled to room temperature, which is generally about 25°C. In some embodiments, the acoustic ECM hydrogel is cooled to about 15°C to about 25°C. Acoustic ECM hydrogels can be cooled to about 23°C to about 27°C. Acoustic ECM hydrogels can be cooled to approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C.

[0078] In some embodiments, the mammalian acoustic ECM hydrogel is thermoreversible, and the hydrogel is in a solid (gel) phase at temperatures below about 37°C and in a liquid (sol) phase at temperatures above about 37°C. The acoustic hydrogel can be manufactured using any of the methods disclosed herein.

[0079] In some embodiments, the storage modulus (G') is about an order of magnitude greater than the loss modulus (G”). In further embodiments, the viscosity of the mammalian acoustic ECM hydrogel decreases with increasing stress at temperatures of about 15 to about 37°C, e.g., about 15, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and / or 36°C. In further embodiments, the viscosity of the mammalian acoustic ECM hydrogel decreases with increasing stress at room temperature and / or about 23°C to about 27°C and / or about 15°C to about 25°C. In one embodiment, the gel-to-sol transition of the acoustic ECM hydrogel occurs at about 37°C, thereby allowing the hydrogel to be used in anal fistulas as it is sufficiently viscous at body temperature. Compositions comprising mammalian acoustic ECM hydrogel and trehalose can be prepared as discussed below.

[0080] In some embodiments, the composition has a storage modulus (G') to loss modulus (G") ratio in the range of about 6:1 to about 12:1 at 37°C. Thus, in some embodiments, the composition has a storage modulus (G') to loss modulus (G") ratio of 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1 at 37°C. In further embodiments, the composition has a storage modulus (G') to loss modulus (G") ratio in the range of about 7:1 to about 11:1 at 37°C, for example, in the range of about 8:1 to about 10:1 at 37°C.

[0081] In further embodiments, the composition has a storage modulus (G') of about 5 to about 15,000 Pa. In even more embodiments, the composition may have a G' of about 5 to about 10,000 Pa, about 5 to about 5,000 Pa, about 5 to about 500 Pa, or about 5 to about 50 Pa. In further embodiments, the composition may have a G' of about 10 to about 15,000 Pa, about 100 to about 15,000 Pa, about 1,000 to about 15,000 Pa, about 2,000 to about 15,000 Pa, about 3,000 to about 15,000 Pa, about 4,000 to about 15,000 Pa, about 5,000 to about 15,000 Pa, about 6,000 to about 15,000 Pa, or about 7,000 It can have G' values ​​in the following ranges: 0 to approximately 15,000 Pa, approximately 8,000 to approximately 15,000 Pa, approximately 9,000 to approximately 15,000 Pa, approximately 10,000 to approximately 15,000 Pa, approximately 11,000 to approximately 15,000 Pa, approximately 12,000 to approximately 15,000 Pa, approximately 13,000 to approximately 15,000 Pa, or approximately 14,000 to approximately 15,000 Pa.

[0082] In some embodiments, the composition is about 0.1 to 1 s -1 It has a viscosity of approximately 0.1 to approximately 0.5 s. -1 Or approximately 0.5 to 1 second -1 It can have a viscosity of about 0.2 to about 0.9 s. - 1, or approximately 0.3 to 0.8 seconds -1 , or approximately 0.4 to 0.7 seconds -1 , or approximately 0.5 to 0.6 seconds -1 It can have a viscosity of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 s. -1 It has a viscosity of [value].

[0083] In some embodiments, a composition comprising mammalian acoustic ECM hydrogel and trehalose is prepared. In further embodiments, the composition comprises about 0.1 mg / ml to about 700 mg / ml of trehalose. In some embodiments, the composition comprises about 1 mg / ml of trehalose to about 700 mg / ml of trehalose. In further embodiments, the composition comprises 50 mg / ml to about 500 mg / ml of trehalose. In other embodiments, the composition comprises about 10 mg / ml to about 600 mg / ml of trehalose, about 10 mg / ml to about 500 mg / ml, about 10 mg / ml to about 400 mg / ml, about 10 mg / ml to about 300 mg / ml, about 10 mg / ml to about 200 mg / ml, or about 10 mg / ml to about 100 mg / ml of trehalose. In further embodiments, the composition may contain about 0.1 to about 100 mg / ml of trehalose, about 0.1 to about 10 mg / ml of trehalose, or about 0.1 to about 1 mg / ml of trehalose. In many embodiments, the composition may contain about 50 mg / ml to about 400 mg / ml of trehalose, about 50 mg / ml to about 300 mg / ml of trehalose, about 50 mg / ml to about 200 mg / ml of trehalose, or about 50 mg / ml to about 100 mg / ml of trehalose. In some embodiments, the composition contains about 20 mg / ml to about 70 mg / ml of trehalose. In some embodiments, the composition contains about 10 mg / ml to about 100 mg / ml of trehalose. In some embodiments, the composition contains 15 to 30 mg / ml of trehalose. In some embodiments, the composition contains 60 to 70 mg / ml of trehalose. In some embodiments, the composition contains 20 mg / ml of trehalose. In some embodiments, the composition contains 66 mg / ml of trehalose. In other embodiments, the composition may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 66, 70, 80, 90, 100, 200, 300, 400, 500, or 600 mg / ml of trehalose.In other embodiments, the composition may contain trehalose in amounts ranging from about 100 mg / ml to about 700 mg / ml, for example, about 100, 150, 20, 250, 300, 350, 400, 450, 500, 550, or 600 mg / ml. In further embodiments, the composition may contain trehalose in amounts ranging from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / ml.

[0084] In further embodiments, the composition comprises a mammalian acoustic ECM hydrogel containing solubilized ECM, additional pulverized mammalian ECM, and optionally trehalose. The pulverized ECM is not treated with ultrasound and is not solubilized in the hydrogel. The pulverized ECM is a separate additive to the composition, which also contains the mammalian ECM hydrogel. The composition may contain about 1 to about 30% (weight per volume (w / v)) of pulverized ECM that has not been solubilized in the acoustic ECM hydrogel. Although not bound by theory, pulverized ECM generally has intact collagen particles, whereas the acoustic ECM hydrogel has collagen that has been destroyed by ultrasound, resulting in an increased soluble collagen content (Hussey et al., Ultrasonic cavitation to prepare ECM). hydrogels Acta Biomater. 2020 May;108:77-86; the whole is incorporated herein by reference (see, for example, Figure 2). Thus, acoustic ECM hydrogel compositions containing further pulverized mammalian ECM include both intact collagen and disrupted collagen.

[0085] The composition may contain approximately 5% to 30% w / v, approximately 10% to 30%, approximately 15% to 30%, approximately 20% to 30%, approximately 25% to 30%, approximately 1% to 20%, approximately 5% to 20%, approximately 10% to 20%, approximately 15% to 20%, approximately 10% to 20%, or approximately 15% to 20% of pulverized ECM (w / v). The composition may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% of pulverized ECM (w / v). The composition may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% or less of pulverized ECM (w / v). The composition may contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% of ground ECM (w / v).

[0086] Grinded ECM can originate from the same species as mammalian acoustic ECM hydrogels. In one specific, non-limiting example, both the mammalian acoustic ECM hydrogel and the grinding ECM are from pigs. In another non-limiting example, both the mammalian acoustic ECM hydrogel and the grinding ECM are from humans.

[0087] The pulverized ECM may originate from the same or different tissue as the mammalian acoustic ECM hydrogel. In one embodiment, the mammalian acoustic ECM hydrogel and the pulverized ECM originate from the same tissue. In one embodiment, the mammalian acoustic ECM hydrogel and the pulverized ECM are dermal ECM. In one embodiment, the mammalian acoustic ECM hydrogel and the pulverized ECM are porcine dermal ECM.

[0088] In a specific, non-limiting example, the composition comprises i) a mammalian acoustic extracellular matrix (ECM) hydrogel, a) the mammalian acoustic ECM hydrogel is thermoreversible, being in the gel phase at temperatures below about 37°C and transitioning to the liquid phase at temperatures above about 37°C, and b) the mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of about 0.1 mg / ml to about 1,000 mg / ml. The composition also comprises i) 0.1 mg / ml to about 700 mg / ml of trehalose. The composition also comprises ii) about 1 to about 30% (by weight per volume) of pulverized ECM that is not solubilized in the hydrogel. In some embodiments, the composition has a storage modulus (G') to loss modulus (G") ratio of about 6:1 to about 12:1 at 37°C.

[0089] In a specific non-limiting example, the composition comprises: i) a mammalian acoustic extracellular matrix (ECM) hydrogel, where a) the mammalian acoustic ECM hydrogel is thermoreversible, being in the gel phase at a temperature below about 37°C and transitioning to the liquid phase at a temperature above about 37°C; b) the mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of about 0.1 mg / ml to about 1,000 mg / ml; c) the composition has a storage modulus (G’) to loss modulus (G”) ratio of about 6:1 to about 12:1 at 37°C. The composition also comprises: ii) trehalose at a concentration of 0.1 mg / ml to about 700 mg / ml. The composition also comprises: iii) about 1 to about 30% (weight per volume) of fragmented ECM that is not solubilized in the hydrogel. In some non-limiting examples, the mammalian acoustic ECM hydrogel contains bladder ECM, small intestinal submucosa ECM, esophageal ECM, tracheal ECM, liver ECM, or skin ECM. In other non-limiting examples, the ECM is porcine ECM. In other non-limiting examples, the ECM is skin ECM. In other non-limiting examples, the ECM is porcine skin ECM. In further non-limiting examples, the mammalian acoustic ECM hydrogel does not contain exogenous protease or inactivated exogenous protease, such as exogenous pepsin, trypsin, or hyaluronidase, or the inactivated form of exogenous pepsin, trypsin, or hyaluronidase. In more non-limiting examples, the composition contains about 50 to about 500 mg / ml of trehalose. In still other non-limiting examples, the composition has a storage modulus (G’) of about 5 to about 15,000 Pa. In additional non-limiting examples, the composition has a viscosity of about 0.1 to 1 s -1 at a temperature of about 25°C and a storage modulus of 5 to 15,000 Pa. In other non-limiting examples, the concentration of mammalian solubilized ECM in the mammalian acoustic ECM hydrogel is about 20 mg / ml to about 70 mg / ml, such as about 40 to about 66 mg / ml. Other aspects of the disclosed composition are provided above.

[0090] The composition may be sterilized before application to the target. The composition may be sterilized using any method known to those skilled in the art, including filtration and radiation. In some embodiments, the composition is sterilized with ionizing radiation such as e-beams or gamma rays. The composition may be sterilized using gamma rays, for example, using irradiation of 10-50 kGy, such as 15-45 kGy, 20-40 kGy, or 10-30 kGy. In some non-limiting examples, the composition is sterilized using irradiation of 10, 15, 20, 25, 30, 35, 40, 45, or 50 kGy. Generally, the composition is sterilized for a time sufficient to achieve the absence of detectable viable pathogens such as viruses and bacteria, but not limited to these.

[0091] Antibiotics or antimicrobial agents can be added to a composition to reduce the likelihood of infection at the treatment site. A variety of antibiotics are known, including those that target bacterial cell walls (e.g., penicillins and cephalosporins) or cell membranes (e.g., polymyxins), or those that interfere with essential bacterial enzymes (e.g., quinolones and sulfonamides). Antibiotics include, but are not limited to, clindamycin, erythromycin, tetracycline, minocycline, doxycycline, penicillin, ampicillin, carbenicillin, methicillin, cephalosporins, vancomycin and bacitracin, streptomycin, gentamicin, chloramphenicol, fusidic acid, ciprofloxacin and other quinolones, sulfonamides, trimethoprim, dapsone, isoniazid, teicoplanin, avoparcin, synacid, virginiamycin, cefotaxime, ceftriaxone, piperacillin, ticarcillin, cefepime, cefpirome, rifampicin, pyrazinamide, ciprofloxacin, levofloxacin, enrofloxacin, amikacin, netylmycin, imipenem, meropenem, inezolid, their pharmaceutically acceptable salts and their prodrugs. Antimicrobial agents also include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), and oxazolidinones (such as linezolid). Antibiotics can be narrow-spectrum or broad-spectrum. Antibiotics can target Gram-negative or Gram-positive bacteria. Topical antibiotics may include active agents such as macrolide antibiotics (such as erythromycin), sulfonamide antibiotics (such as sulfacetamide), cyclic peptides (such as bacitracin and polymyxin), pseudodomonic acid (such as mupirocin), aminoglycosides (such as neomycin), or quinolones (such as ciprofloxacin or ofloxacin), nitroimidazoles (such as metronidazole), or combinations of drugs (such as bacitracin / polymyxin or neomycin / polymyxin B / bacitracin).

[0092] Furthermore, local anesthetics such as lidocaine may be added to the composition to minimize discomfort. Any suitable additives can be used, as long as they are appropriate for the composition and the specific patient and disease condition being treated.

[0093] In some embodiments, compositions such as sterile compositions can be injected through a 5Fr / 16G catheter. In one embodiment, the composition can be injected through a 5Fr / 16G catheter at room temperature, or at both room temperature and approximately 37°C.

[0094] Any useful agent can be mixed with, co-delivered, co-administered, or otherwise combined with any composition described herein. Examples of useful agents, but not limited to, include interferons, interleukins, chemokines, cytokines, hormones, coagulants, chemotherapeutic agents, and antibiotics. Enzyme ECM hydrogel and composition for use

[0095] Disclosed herein are compositions that can be administered topically to fistulas, such as anal fistulas, and thus can be used in the treatment of fistulas, for example, to close the fistulas. The compositions comprise a mammalian enzyme extracellular matrix (ECM) hydrogel that is thermoreversible, exists in a gel phase at temperatures above 37°C, and transitions to a liquid phase at temperatures below approximately 37°C. The enzyme-prepared ECM hydrogel is discussed, for example, in U.S. Patent No. 8,361,503, which is incorporated herein by reference in its entirety for all purposes. The preparation of the finally sterile enzyme-prepared ECM hydrogel is discussed, for example, in U.S. Patent No. 10,213,526, which is incorporated herein by reference in its entirety for all purposes.

[0096] An "enzymatic ECM hydrogel" refers to a hydrogel composed of extracellular matrix in which the extracellular matrix (ECM) is enzymatically digested. For example, the ECM can be digested by proteases, such as acidic proteases like trypsin or pepsin.

[0097] Enzymatic ECM hydrogels can be made from any mammalian ECM source tissue or organ, including, but not limited to, the bladder, intestines (small or large intestine, etc.), heart, kidneys, uterus, brain, blood vessels, lungs, bones, muscles, pancreas, stomach, spleen, adipose tissue, muscle tissue, liver, esophagus, placenta, and dermis. The source of the ECM may be, for example, a pig, a cattle, a human, or a sheep. In a specific non-limiting example, the ECM is porcine ECM. In another non-limiting example, the ECM is bladder ECM, small intestine submucosal ECM, esophageal ECM, tracheal ECM, liver ECM, or cutaneous ECM. In one embodiment, the ECM is bladder ECM. In another embodiment, the ECM is cutaneous ECM. In yet another embodiment, the ECM is small intestine submucosal ECM.

[0098] Enzymatic ECM hydrogels are produced by enzymatic digestion of the extracellular matrix under specific conditions. For example, a method for preparing an enzymatic extracellular matrix-derived gel is provided. In one embodiment, the method comprises (i) pulverizing the extracellular matrix, (ii) solubilizing the intact, non-dialysis, or non-crosslinked extracellular matrix by digestion with an acidic protease in an acidic solution to produce a digested solution, (iii) raising the pH of the digested solution to 7.2–7.8 to produce a neutralized digested solution, and (iv) gelling the neutralized digested solution at a temperature above approximately 25°C.

[0099] In one non-limiting embodiment, the ECM is freeze-dried, pulverized, and then solubilized with an acidic protease. The acidic protease may be pepsin or trypsin, but is not limited to pepsin. In one embodiment, the ECM is solubilized in a solution with an acidic pH suitable for or optimal for the protease, e.g., above pH 2, or between pH 2 and 4, e.g., a 0.01 M HCl solution. The solution is typically solubilized for 12 to 48 hours with mixing, depending on the type of tissue (e.g., stirring, mixing, blending, rotating, inversion).

[0100] Once the ECM is solubilized (typically substantially completely), the pH is raised to 7.2–7.8, and according to one embodiment, to pH 7.4. The pH of the solution can be increased using a base, such as a base containing hydroxyl ions including NaOH. Similarly, a buffer, such as an isotonic buffer including but not limited to phosphate-buffered saline (PBS), can be used to bring the solution to a target pH or to help maintain the pH and ionic strength of the gel to a target level, such as physiological pH and ionic conditions. The neutralized digested solution can gel at temperatures close to 37°C, typically any temperature above 25°C, but gelation proceeds much more rapidly at temperatures above 30°C and as the temperature approaches physiological temperature (i.e., 37°C). This method typically does not involve a pre-gelling dialysis step and typically results in a more complete ECM-like matrix that gels more slowly at 37°C than comparable collagen or dialysis ECM preparations.

[0101] In one embodiment, the enzyme ECM hydrogel is subjected to final sterilization. "Final sterilization" of the enzyme ECM hydrogel refers to the essential or substantially complete sterilization of the composition. Final sterilization does not include, for example, disinfection with peracetic acid during the preparation of the ECM product as part of or associated with the decellularization of the ECM.

[0102] In one embodiment, the final sterile enzymatic ECM hydrogel is prepared by (i) pulverizing the extracellular matrix, (ii) solubilizing intact, non-dialysis-free, or non-crosslinked extracellular matrix by digestion with an acidic protease in an acidic solution to produce a digestate, (iii) drying the digestate to produce a dried digest, (iv) finally sterilizing the dried digest to produce a sterile dried digest, (iv) hydrating the sterile dried digest to produce a sterile digestate, and (v) raising the pH of the sterile digestate to 7.2–7.8 to produce a neutralized sterile digestate. The neutralized and sterile digestate is sometimes called a "pregel." The neutralized digestate or pregel can then be gelled by raising the temperature of the neutralized digestate to a temperature higher than approximately 25°C.

[0103] Final sterilization of dried digests can be achieved, for example, by exposure to an electron beam or gamma ray, ethylene oxide gas, or supercritical carbon dioxide.

[0104] The drying of the digested solution can be achieved, for example, by air drying, freeze-drying, or heating. “Drying,” “to dry,” or “dried” means drying or freeze-drying to the point where substantially all water is removed from the composition, and it should be recognized that in practice it is not possible to literally remove all water molecules from any composition. Therefore, “drying” or “dried” refers to, for example, a water content of less than 5.0, 1.0, 0.5, 0.1, 0.01, 0.001, or 0.0001% (by weight) of the composition, but not limited to these. The material can be dried by any process, for example, by simple evaporation at any non-damaging temperature such as room temperature, or by freeze-drying.

[0105] Hydration of sterile dried digests can be achieved, for example, by solubilizing them in sterile water, an aqueous solution such as TRIS buffer or PBS, or a salt solution such as a sodium chloride solution (0.9%) physiological saline to produce a sterile digest solution.

[0106] Neutralizing a hydrated digested solution can be achieved, for example, by mixing the solution with an isotonic buffer or a base, such as, but not limited to, NaOH. Therefore, in one embodiment, the present invention provides a gellable extracellular matrix (ECM) composition comprising a decellularized, enzymatically digested, dried, and final-sterilized intact extracellular matrix, which can form a gel when hydrated, neutralized to pH 7.2-7.8, and heated to a temperature above 25°C for use in repairing fistulas. For example, the composition is used to fill fistula tracts, for example, in humans. In one embodiment, the composition comprises an inactivated protease, such as trypsin or pepsin. For example, the fistula is an anal fistula. In one embodiment, the composition forms a gel when heated to 37°C.

[0107] Accordingly, in one embodiment, the present invention provides a final sterile extracellular matrix (ECM) digestion solution containing decellularized, enzymatically digested, and final sterile intact extracellular matrix, the composition which can form a gel when neutralized to pH 7.2-7.8 and heated to a temperature above 25°C for use in repairing fistulas. For example, the composition is used to fill fistula tracts, for example, in humans. In one embodiment, the composition contains an inactivated protease, for example, trypsin or pepsin. For example, the fistula is an anal fistula. In one embodiment, the composition contains an inactivated protease, for example, trypsin or pepsin. In one embodiment, the composition forms a gel when heated to 37°C. In one embodiment, the solution is an acidic solution.

[0108] Accordingly, in one embodiment, the present invention provides a sterile extracellular matrix (ECM) digestion solution containing hydrated, decellularized, enzymatically digested, dried, and sterile intact extracellular matrix, the digestion solution having a pH of 7.2-7.8 and capable of forming a gel when heated to a temperature above 25°C for use in repairing fistulas. For example, the composition is used to fill fistula tracts, for example, in humans. In one embodiment, the composition contains an inactivated protease, for example, trypsin or pepsin. For example, the fistula is an anal fistula. In one embodiment, the composition contains an inactivated protease, for example, trypsin or pepsin. In one embodiment, the composition forms a gel when heated to 37°C. How to use

[0109] The compositions of this disclosure can be used to treat fistulas in a subject. For example, in one non-limiting embodiment, the fistula is an anal fistula. Most anal fistulas are primary, i.e., they result from a nonspecific infection from the anal glands following the formation of a perianal abscess.

[0110] An anal fistula is a chronic infection duct that communicates the perianal skin with the rectum / anal canal, consisting of an internal opening, a fistula, and an external opening. Anal fistulas clinically manifest as recurrent perianal infections, ulceration, and pus discharge, and perianal cancer may develop in patients who have not been cured for a long period. Traditional treatment methods include fistula excision, fistula incision, suture therapy, fistula opening and drainage, and trans-anorectal mucosa flap internal orifice repair.

[0111] The examples described herein relate to the repair (e.g., closure) of lumens in patients with anal fistulas and other types of fistulas. In particular, the examples described herein involve the use of compositions disclosed herein, formulated for delivery to lumens such as fistulas. Subjects may be any subjects, including veterinary subjects or human subjects. Human subjects may be of any age, including adults and children.

[0112] In some embodiments, the compositions disclosed herein promote tissue growth across the fistula to provide permanent closure. Thus, in some embodiments, the disclosed compositions fill the lumen of any fistula.

[0113] A fistula that follows a straight path from a primary opening to a secondary opening is known as a simple fistula. A fistula containing multiple branching channels from a primary opening and having multiple secondary openings is known as a complex fistula. The disclosed method can be used to treat both simple and complex fistulas.

[0114] In some embodiments, subjects having a fistula are selected for treatment. Fistulas include enterocutaneous fistulas (intestine to skin), colonocutaneous fistulas (colon to skin), enteroenteral fistulas (intestine to intestine), vesicocoli fistulas (bladder to intestine), vesicocoli fistulas (bladder to colon), vesicorectal fistulas (bladder to rectum), rectovaginal fistulas (vagina to rectum), vesicovagina (vagina to bladder), rectoutervagina (uterus to intestine / rectum), vesicouterus (uterus to bladder), ureterovagina (ureter to vagina), retroperitoneal fistulas (uterus to abdominal cavity), or enterovagina (intestine to vagina). In some examples, the disclosed compositions may be used to treat rectovaginal fistulas. Fistulas can be anorectal, rectovaginal, enteric, tracheoesophageal, biliary-intestinal, bladder-vaginal, bladder-intestinal, entero-enteral, pancreatic, cryptoglandular, Crohn's disease, dural venous sinus, colon-bladder, colon-intestinal, colon-vaginal, colon-intestinal, rectourethral, ​​or pharyngeal-cutaneous fistulas.

[0115] A fistula may be an anal fistula. The goal of surgical repair of an anal fistula may be to close the fistula with as little impact as possible on the sphincter muscle. In some situations, the compositions described herein can be delivered locally into the fistula, such as by injection into the lumen. In some embodiments, tissue growth is promoted across the lumen of the anal fistula. Anal fistulas can be simple or complex.

[0116] The anatomical pathways of anorectal fistulas are classified according to their relationship with the anal sphincter. The anal sphincter includes two concentric muscular bands: the medial or internal sphincter and the lateral or external anal sphincter. A fistula passing between the two concentric anal sphincters is known as an intersphincteric fistula. A fistula passing through both the internal and external sphincters is known as a transsphincteric fistula, and a fistula passing over both sphincters is called a suprasphincteric fistula. Fistulas resulting from Crohn's disease usually disregard these anatomical pathways and are known as extrasphincteric fistulas. In one complex type of fistula, the infection begins in the anal gland (primary opening), and two fistulas pass circumferentially around the anal canal, forming a characteristic horseshoe shape. The methods disclosed can be used to treat all of these types of fistulas.

[0117] Methods for injecting a hardening agent or fibrin glue into the tubule of a fistula have evolved. Any of these methods (see, for example, U.S. Patent No. 5,752,974, incorporated herein by reference) can be used with the compositions disclosed herein. The disclosed compositions can be applied by injection, by syringe, by endoscopy, or by catheter. In some embodiments, the disclosed compositions are applied to fill a fistula. Exemplary amounts are 1 ml to 5 ml, but those skilled in the art, such as clinicians, can easily determine an appropriate amount based on clinical parameters such as the diameter and length of the fistula, and / or the method of administration, such as via catheter or endoscopy.

[0118] In some embodiments, preliminary endoscopic visualization (fistuloscopy) and "flushing" of the fistula tract are performed. This procedure can be carried out by a very thin flexible endoscope that is inserted into the secondary opening of the fistula tract and advances through the fistula tract under direct vision, exiting through the primary opening. By performing preliminary fistuloscopy of the fistula tract, the primary opening is accurately identified and the tract is "cleaned out" with the irrigation fluid. Thus, any inflammatory or necrotic tissue in the tract is removed. Following this procedure, an application device such as a syringe, catheter, or endoscope is inserted to allow application of the disclosed composition. In some embodiments, the disclosed composition is applied to fill the fistula. For example, a hydrogel composition is injected into the fistula to fill it from end to end. Since the hydrogel is in a gel state at body temperature, it remains in the fistula tract and is absorbed into the body as the fistula heals. Persons skilled in the art, such as physicians, can easily determine the method of administration of the disclosed composition and evaluate the outcome of the procedure.

[0119] In certain non-limiting cases, the subject may receive antibiotics before use of the disclosed composition. Suitable antibiotics include, but are not limited to, cefuroxime and / or ornidazole. In one embodiment, the subject is anesthetized. Suitable anesthesia procedures include, but are not limited to, spinal anesthesia. Under spinal anesthesia in the prone jackknife position, the fistula is probed to determine the external and internal fistula openings. An anal retractor may be used.

[0120] In some embodiments, the duct is scraped with polyester tape (e.g., white braided fiber, 1 / 8 inch wide). The duct can be cleaned using a blunt tweezers or gauze strips. In some embodiments, the duct is excised to remove granulation tissue, etc., and then cleaned with a solution such as phosphate-buffered saline. Since the composition is injected into the fistula through an external opening, the tip of the injection device is visible protruding from the internal opening into the anal canal. In some embodiments, a tube or other injection device is introduced into the bottom of the fistula-in-ano and then continuously withdrawn during injection to completely fill the fistula with the composition. After the composition has been introduced, any anal retractors can be removed. Postoperative analgesics may be used. In some embodiments, perioperative oral intake is restricted, for example, for about 24 hours. For about 1 to 2 days after the procedure, the patient can be given a liquid diet, and then gradually progressed to a normal diet. A physical examination may be performed.

[0121] While the examples described herein are discussed in relation to anorectal fistulas, it should be understood that the following exemplary devices and techniques can be readily applied to various other types of fistulas, including but not limited to enterocutaneous fistulas (intestine to skin), cocolocutaneous fistulas (colon to skin), enteroenteral fistulas (intestine to intestine), vesicocoli fistulas (bladder to intestine), vesicocolonic fistulas (bladder to colon), vesicorectal fistulas (bladder to rectum), rectovaginal fistulas (vagina to rectum), vesicovaginal fistulas (vagina to bladder), rectouteritus fistulas (uterus to intestine / rectum), vesicouterine fistulas (uterus to bladder), ureterovaginal fistulas (ureter to vagina), retroperitoneal fistulas (uterus to abdominal cavity), or enterovaginal fistulas (intestine to vagina), and tracheoesophageal fistulas.

[0122] In some embodiments, the use of the disclosed composition results in the healing of anal fistulas. Thus, the fistula can be sealed immediately after application. In other embodiments, the use of the disclosed composition promotes tissue growth that seals the fistula over several days, weeks, or months after the use of the composition. In yet another embodiment, tissue growth can be induced within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the use of the disclosed composition. With the disclosed composition, tissue growth can also be induced within 1, 2, 3, or 4 weeks after use. In some embodiments, the composition is absorbed into the body and replaced by tissue growth, resulting in the fistula being replaced by tissue, and the composition no longer being present after several days or weeks.

[0123] In further embodiments, the use of the disclosed composition modulates macrophages toward a remodeling-promoting phenotype (Fizz-1+). Thus, in some embodiments, the use of the disclosed composition increases the number of Fizz-1+ macrophages at the site of anal fistula. [Examples]

[0124] Disclosed is a space-filling, remodeling-promoting colloidal hydrogel derived from decellularized extracellular matrix (ECM) that can be used to treat fistulas such as anorectal fistulas or any other fistulas disclosed herein. ECM derived from porcine dermis (dECM), etc., was exposed to sonication in the presence of physiological buffer, supplemented with further dECM powder ("colloid") and trehalose, and cooled to form an injectable hydrogel. In vitro results demonstrate that this type of ECM hydrogel is a rigid biomaterial with hemostatic properties that can fill fistula tracts with complex structures at body temperature, can regulate macrophages toward a remodeling-promoting phenotype (Fizz+), and has hemostatic properties. The hydrogel maintained its rigidity in the ex vivo tract, did not degrade at body temperature, and did not leak from the tract.

[0125] In a postmortem ex-vivo pig fistula model, the biomaterial was able to fill the duct without leakage at body temperature. The space-filling, anti-inflammatory, and hemostatic capabilities of the colloidal ECM hydrogel make it effective for treating simple and complex anorectal fistulas. The ease of injection, sterility, and stability of the biomaterial at both room temperature and body temperature allow it to be used "off-the-shelf" by physicians in both surgical and outpatient settings. The ECM colloidal hydrogel can improve healing, mitigate surgical bleeding, and reduce complications and recurrence in the management of anorectal fistulas. Example 1 Hydrogel Stability

[0126] It has been previously shown that widely accepted final sterilization methods (i.e., gamma irradiation, electron beam irradiation, and ethylene oxide (EtO) exposure) inhibit the formation of ECM hydrogels prepared using pepsin digestion (White et al., Front.Bioeng.Biotechnol.Conference Abstract:10th World Biomaterials Congress.doi:10.3389 / conf.FBIOE.2016.01.00032, and White et al., Journal of Immunology and Regenerative Medicine.2018;2:11-20). Therefore, we conducted a study to determine whether final sterilization inhibits the gelation of ECM hydrogels prepared using sonication. This example shows that the addition of trehalose to the acoustic hydrogel material before sterilization helps preserve the properties of the hydrogel after sterilization.

[0127] As shown in Figure 1 (left panel), sterilization of the acoustic gel with a 35 kGy E-beam resulted in the formation of a series of aggregates rather than uniform consistency. However, the addition of either 20 or 40 mg / ml of trehalose prevented the acoustic ECM hydrogel from forming aggregates after sterilization with a 35 kGy E-beam (Figure 1 right panel), and instead resulted in uniform consistency.

[0128] Next, it was determined whether trehalose could prevent the formation of aggregates in colloidal acoustic hydrogels. Subsequently, 100 mg / ml of cutaneous ECM was solubilized using ultrasonic cavitation, and then further pulverized cutaneous ECM was added to the suspension as a thickener to produce colloidal ECM hydrogels ranging from 0.1 to 20% colloid (w / v). As shown in Figure 2, 5% (w / v) colloidal acoustic gels prepared with 40 or 66 mg / ml of trehalose were sterilized using gradually increasing dose E-beam irradiation. After sterilization, the samples were cooled to 4°C and injected into ring molds. Macroscopic evaluation showed that, when injected into ring molds at 4°C, the 5% colloidal hydrogel containing 40 mg / ml trehalose formed more aggregates than the dECM colloidal hydrogel containing 66 mg / ml trehalose at all E-beam doses tested.

[0129] In another experimental setup, the sample was cooled to 4°C, injected into a ring mold, then placed in a 37°C incubator for 1 hour, and the ring mold was then carefully removed to determine whether the colloidal gel could retain its shape after E-beam sterilization (Figure 3).

[0130] The results showed that samples containing 66 mg / ml trehalose maintained their shape better than samples prepared with 40 mg / ml trehalose at all E-beam doses tested. Furthermore, gentle handling of the samples (by pressing down the central gel) showed that dECM colloidal hydrogels containing 40 mg / ml trehalose decomposed more easily under gentle handling conditions than colloidal hydrogels containing 66 mg / ml trehalose (Figure 4).

[0131] Next, the viscoelastic properties of dermal ECM colloidal hydrogels containing trehalose (Figures 5A-5B) were determined. 2, 5, or 10% colloidal hydrogels (w / v) containing 20 or 40 mg / ml trehalose were subjected to E-beam sterilization using a dose of 25 kGy. The average storage modulus of the samples is shown in Figure 5A. The results showed that the sample containing 20 mg / ml trehalose was stiffer than the sample containing 40 mg / ml trehalose, indicating that increasing the colloidal concentration increased the stiffness of the material.

[0132] Finally, the biological activity of trehalose against mouse bone marrow-derived macrophages was evaluated (Figure 6). The assay results showed that trehalose failed to induce iNOS or Fizz1 expression compared to the control. In summary, the data indicate that trehalose can be used as an excipient in the preparation of acoustic gels and colloidal acoustic gels due to its inert biological activity and its ability to reduce stiffness and prevent aggregate formation in colloidal acoustic ECM hydrogels after E-beam sterilization, resulting in a uniform consistency of the hydrogel. Example 2 material and method

[0133] Preparation of cutaneous ECM: The cutaneous ECM was prepared as described above (Reing JE, et al. Biomaterials. 2010;31(33):8626-33). Briefly, full-thickness skin was harvested from a market-weight (approximately 110 kg) pig (Tissue Source Inc.), and the subcutaneous fat and epidermis were removed by mechanical delamination. This tissue was then treated with 0.25% trypsin (Thermo Fisher Scientific) for 6 hours, 70% ethanol for 10 hours, 3% H2O2 for 15 minutes, and 1% Triton® X-100 (Sigma-Aldrich) in 0.26% EDTA / 0.69% tris for 6 hours, with the solutions changed for a further 16 hours, and treated with 0.1% peracetic acid / 4% ethanol (Rochester Midland) for 2 hours. Washing with water was performed between each chemical change and with alternating washes of water and phosphate-buffered saline (PBS) after the final step. All chemical exposures were carried out at 300 rpm with stirring on an orbital shaker. The skin ECM was then freeze-dried and ground into fine particles using a Wiley Mill equipped with a #60 mesh screen.

[0134] Preparation of colloidal ECM hydrogels: 100 mg of ECM powder was resuspended in phosphate-buffered saline (PBS) in a 50 mL conical tube with or without trehalose, and sonicated at 100% amplitude for 5 minutes using a FISHERBRAND® Model 120 Sonic Dismembrator equipped with a 1 / 8” probe. After sonication, the solubilized ECM solution was mixed with further ECM powder to prepare 2, 5, or 10% (w / v) colloidal gel suspensions. After mixing the suspensions, the samples were placed in 3 ml syringes and incubated at 4°C to induce gelation.

[0135] Macrophage activation: Mouse bone marrow was collected from 6-8 week old B6 mice. Cells collected from the bone marrow were washed and 2 × 10⁶ cells were extracted. 6Cells were seeded at a concentration of cells / mL and differentiated into macrophages for 7 days in the presence of macrophage colony-stimulating factor (MCSF), with complete medium changes every 48 hours. Macrophages were then activated for 24 hours with one of the following: 1) 20 ng / mL interferon-γ (IFNγ) and 100 ng / mL lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, California; Sigma Aldrich). IFNγ+LPS 1) The phenotype (M1-like) was promoted; 2) M was induced by 20 ng / mL interleukin (IL)-4 (Invitrogen). IL-4 The phenotype (M2-like) was promoted by: 3) gradually increasing concentrations of trehalose (25, 50, 100, 200 mM), or 4) 5 mg / ml UBM acoustic gel. After a 24-hour incubation period at 37°C, the cells were washed with sterile PBS and fixed with 2% paraformaldehyde (PFA) for immunolabeling. To prevent nonspecific binding, the cells were incubated at room temperature for 1 hour in a blocking solution consisting of PBS, 0.1% Triton®-X, 0.1% Tween®-20, 4% goat serum, and 2% bovine serum albumin. The blocking buffer was then removed, and the cells were incubated with primary antibody. The cells were incubated at 4°C for 16 hours, the primary antibody was removed, and the cells were washed with PBS. A solution of fluorophore-complexed secondary antibody was added to the wells at room temperature for 1 hour. The antibody was then removed, the cells were washed with PBS, and the nuclei were counterstained using DAPI. Cytokine-activated macrophages were used to establish a standardized exposure time (positive control), which was then maintained constant across the entire group. Example 3 Repair of anal fistula

[0136] Current standard treatments for anal, rectal, and enterocutaneous fistulas remain ineffective, associated with high complication rates, and present a persistent clinical challenge. Surgical approaches to fistula treatment, such as advancement flaps, are associated with high fecal incontinence rates and poor outcomes. Alternatively, sphincter-preserving methods, such as fistula plugs, are associated with widely varying clinical outcomes due to plug extrusion, a major failure mechanism, and are often incompatible with complex fistula structures, requiring multiple interventions and prolonged draining seton placement. Currently, there are no available treatment options that preserve sphincter muscle tissue, reduce complication and failure rates, accommodate complex fistulas, and promote timely healing of the fistula tract. Extracellular matrix (ECM)-based shear-reducing hydrogel formulations may be a better solution to this unmet clinical need. Using ECM hydrogels and colloidal ECM hydrogels prepared using ultrasonic cavitation, fistula tracts can be filled intersphincteric, transsphincteric, supersphincteric, extrasphincteric, or submucosal, thereby enhancing and facilitating tract closure, even in cases of complex and multi-tubular fistulas, by enabling timely host tissue integration without fecal incontinence or extrusion. ECM hydrogels can be injected into the fistula tract after debridement of the tract, and a drainage seton is used preoperatively. Rheological analysis has shown that when ECM hydrogels and colloidal ECM hydrogels are readily administered via catheter, they remain in situ, enabling effective and timely repair of fistulas by allowing host tissue integration and ultimately tract closure. ECM hydrogels are intended to degrade within 7–120 days and be replaced by new host tissue.

[0137] As shown in Figures 7A-D, sterile acoustic ECM hydrogel can be introduced into the tract of an anal fistula using a catheter. In this case, the catheter is introduced from the external opening on the anal side of the fistula. The catheter is inserted through the fistula to the internal opening, and then the catheter is retracted to deposit the acoustic ECM hydrogel composition into the tract. Once the tract is filled, the catheter is removed. The acoustic ECM hydrogel remains as a gel in the tract at body temperature and does not leak out of the tract. This is shown in Figure 7E, which is a photograph of a pig with an anorectal fistula filled with acoustic ECM hydrogel, indicated as a dark circle at approximately 11 o'clock in the anus. Furthermore, due to its physical properties at body temperature, it retains sufficient rigidity to withstand the forces applied to the anorectal region during sitting and defecation.

[0138] For each surgery, the pigs were placed in the non-penetrating position (NPO). Intestinal contents were removed 12 hours preoperatively. The pigs were sedated with intramuscular injections of ketamine / xylazine (20 mg / kg (K) and 2 mg / kg (XY)) and terazole. Anesthesia was maintained throughout the surgery with continuous isofluorane. The pigs were placed in the supine position in the dorsal lithotomy position. Using an 11-blade scalpel, incisions were made in the ischial anal fossa for a total of four fistulas at the 2-, 5-, 8-, and 11 o'clock positions. Using curved hemostatic forceps, blunt incisions were made through the muscular tissue of the anal sphincter to the dentate line of the anal mucosa. A 1 mm incision was made at the dentate line to connect the drainage channels. Fourteen French silicone drainage setons were inserted into the fistulas, tied, and sutured to the skin to prevent movement. The setons were maintained for 4 weeks postoperatively to establish patency.

[0139] Four weeks later, the animals were sedated, placed in the dorsal lithotomy position, the seton was removed, and the tubule was cleaned with continuous washing of 70% EtOH and iodine scrub. The tubule was filled with dECM colloidal gel, and the internal opening of the fistula was closed with a single 4-0 figure-eight suture. The external opening was left open to allow for gel drainage.

[0140] In the case of fistula repair, it may be possible to close one end of the tract with one or more sutures before injecting the ECM acoustic hydrogel. For example, if there is an opening larger than the catheter, sutures can be placed to create a pocket that will be filled with hydrogel, for example, at the end that will be filled first. Furthermore, one or more sutures can be placed at each end of the fistula to close the tissue around the hydrogel. This may be suitable for situations where the fistula is wide and not very long.

[0141] Considering the many possible embodiments to which the principles of the present invention may be applied, it should be recognized that the illustrated embodiments are merely examples of the present invention and should not be considered limitations on the scope of the invention. Rather, the scope of the present invention is defined by the following claims. Accordingly, the inventors claim as their invention everything that falls within the scope of these claims and spirit. The present invention provides, for example, the following items: (Item 1) A method for treating a fistula in a subject, The method comprises topically administering an effective amount of a composition containing a mammalian acoustic extracellular matrix (ECM) hydrogel to the fistula in the subject. a) The mammalian acoustic ECM hydrogel is thermoreversible, and the mammalian acoustic ECM hydrogel is in the gel phase at temperatures below approximately 37°C and transitions to the liquid phase at temperatures above approximately 37°C. b) The mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of approximately 0.1 mg / ml to approximately 1,000 mg / ml. c) The composition has a storage modulus (G') to loss modulus (G") ratio in the range of about 6:1 to about 12:1 at 37°C, A method for treating the fistula in the subject. (Item 2) The method according to item 1, wherein the composition further comprises a radioprotective agent, the radioprotective agent comprising 0.1 mg / ml to about 700 mg / ml of trehalose. (Item 3) The method according to item 1 or item 2, further comprising sterilizing the composition with ionizing radiation. (Item 4) The method according to item 3, wherein the ionizing radiation is e-beam or gamma-ray irradiation. (Item 5) The method according to any one of items 3 to 4, wherein the composition is sterilized using irradiation of 10 to 50 kGy. (Item 6) The method according to any one of items 3 to 5, wherein the composition is irradiated with gamma rays. (Item 7) The method according to any one of items 1 to 6, wherein the mammalian acoustic ECM hydrogel includes a bladder ECM, a small intestinal submucosa (SIS) ECM, an esophageal ECM, a tracheal ECM, a liver ECM, or a cutaneous ECM. (Item 8) The method according to any one of items 1 to 7, wherein the mammalian acoustic ECM hydrogel comprises porcine ECM. (Item 9) The method according to any one of items 1 to 8, wherein the mammalian acoustic ECM hydrogel does not contain an exogenous protease or an inactivated exogenous protease. (Item 10) The method according to any one of items 1 to 9, wherein the mammalian acoustic ECM hydrogel does not contain exogenous pepsin, trypsin, or hyaluronidase, or an inactivated form of exogenous pepsin, trypsin, or hyaluronidase. (Item 11) The method described in any one of items 1 to 10, wherein the subject is a human. (Item 12) The method according to any one of items 1 to 11, wherein the composition is injectable through a 5Fr / 16G catheter. (Item 13) The method according to any one of items 2 to 12, wherein the composition comprises about 50 to about 500 mg / ml of trehalose. (Item 14) The method according to any one of items 1 to 13, wherein the composition further comprises about 1 to about 30% (by weight per volume) of pulverized ECM that has not been solubilized in the hydrogel. (Item 15) The method according to any one of items 1 to 14, wherein the composition has a storage modulus (G') of about 5 to about 15,000 Pa. (Item 16) The composition is heated at a temperature of approximately 25°C for approximately 0.1 to 1 second. -1 The method according to any one of items 1 to 15, having a viscosity and a storage modulus of 5 to 15,000 Pa. (Item 17) The method according to any one of items 1 to 16, wherein the concentration of the solubilized ECM in the mammalian acoustic ECM hydrogel is approximately 20 mg / ml to approximately 70 mg / ml. (Item 18) The method according to any one of items 1 to 17, wherein the concentration of the solubilized ECM in the mammalian acoustic ECM hydrogel is about 40 to about 66 mg / ml. (Item 19) The method according to any one of items 1 to 18, wherein the fistula is an anal fistula, and the anal fistula is an intersphincteric anal fistula, a transsphincteric anal fistula, a suprasphincteric anal fistula, an extrasphincteric anal fistula, or a submucosal anal fistula. (Item 20) The method according to any one of items 1 to 19, wherein the composition is administered to fill the duct of the fistula. (Item 21) A composition, i) Mammalian acoustic extracellular matrix (ECM) hydrogel, a) The mammalian acoustic ECM hydrogel is thermoreversible, and the mammalian acoustic ECM hydrogel is in the gel phase at temperatures below approximately 37°C and transitions to the liquid phase at temperatures above approximately 37°C. b) The mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of approximately 0.1 mg / ml to approximately 1,000 mg / ml. c) The composition comprises a mammalian acoustic ECM hydrogel having a storage modulus (G') to loss modulus (G") ratio of about 6:1 to about 12:1 at 37°C, ii) Trehalose in concentrations of 0.1 mg / ml to approximately 700 mg / ml, iii) Approximately 1 to approximately 30% (by weight per volume) of pulverized ECM that has not been solubilized in the hydrogel, A composition containing the following: (Item 22) The composition according to item 21, wherein the composition has been irradiated with gamma rays. (Item 23) The composition according to item 21 or 22, wherein the mammalian acoustic ECM hydrogel comprises a bladder ECM, a small intestinal submucosal ECM, an esophageal ECM, a tracheal ECM, a liver ECM, or a cutaneous ECM. (Item 24) The composition according to any one of items 21 to 23, wherein the aforementioned ECM includes porcine ECM. (Item 25) The composition according to any one of items 21 to 24, wherein the mammalian acoustic ECM hydrogel does not contain an exogenous protease or an inactivated exogenous protease. (Item 26) The composition according to any one of items 21 to 25, wherein the mammalian acoustic ECM hydrogel does not contain exogenous pepsin, trypsin, or hyaluronidase, or an inactivated form of exogenous pepsin, trypsin, or hyaluronidase. (Item 27) The composition according to any one of items 21 to 26, wherein the composition is injectable through a 5Fr / 16G catheter. (Item 28) The composition according to any one of items 21 to 27, wherein the composition comprises about 50 to about 500 mg / ml of trehalose. (Item 29) The composition according to any one of items 21 to 28, wherein the composition has a storage modulus (G') of about 5 to about 15,000 Pa. (Item 30) The composition is heated at a temperature of approximately 25°C for approximately 0.1 to 1 second. -1 A composition according to any one of items 21 to 29, having a viscosity and a storage modulus of 5 to 15,000 Pa. (Item 31) The composition according to any one of items 21 to 30, wherein the concentration of the mammalian solubilized ECM in the mammalian acoustic ECM hydrogel is about 20 mg / ml to about 70 mg / ml. (Item 32) The composition according to any one of items 21 to 31, wherein the concentration of solubilized ECM in the mammalian acoustic ECM hydrogel is about 40 to about 66 mg / ml. (Item 33) A composition according to any one of items 21 to 32 for use in the treatment of fistulas in the subject. (Item 34) A composition comprising a mammalian acoustic extracellular matrix (ECM) hydrogel for use in the treatment of fistulas in a subject, a) The mammalian acoustic ECM hydrogel is thermoreversible, and the mammalian acoustic ECM hydrogel is in the gel phase at temperatures below approximately 37°C and transitions to the liquid phase at temperatures above approximately 37°C. b) The mammalian acoustic ECM hydrogel contains solubilized ECM at a concentration of approximately 0.1 mg / ml to approximately 1,000 mg / ml. c) A composition having a storage modulus (G') to loss modulus (G") ratio of about 6:1 to about 12:1 at 37°C. (Item 35) The composition according to item 33, further comprising a radioprotective agent, wherein the radioprotective agent comprises 0.1 mg / ml to about 700 mg / ml of trehalose. (Item 36) The composition according to item 34 or 35, wherein the composition is sterilized using irradiation of 10 to 50 kGy. (Item 37) The composition according to any one of items 33 to 36, wherein the mammalian ECM hydrogel is irradiated with gamma rays. (Item 38) The composition according to any one of items 34 to 37, wherein the solubilized ECM includes bladder ECM, submucosal (SIS) ECM of the small intestine, esophageal ECM, tracheal ECM, hepatic ECM, or cutaneous ECM. (Item 39) The composition according to any one of items 34 to 38, wherein the solubilized ECM comprises porcine ECM. (Item 40) The composition according to any one of items 34 to 39, wherein the mammalian acoustic ECM hydrogel does not contain an exogenous protease or an inactivated exogenous protease. (Item 41) The composition according to any one of items 33 to 40, wherein the mammalian acoustic ECM hydrogel does not contain exogenous pepsin, trypsin, or hyaluronidase, or an inactivated form of exogenous pepsin, trypsin, or hyaluronidase. (Item 42) The composition according to any one of items 34 to 41, wherein the subject is a human. (Item 43) The composition according to any one of items 34 to 42, wherein the composition is injectable through a 5Fr / 16G catheter. (Item 44) A composition according to any one of items 34 to 43, comprising approximately 50 to approximately 500 mg / ml of trehalose. (Item 45) The composition according to any one of items 34 to 44, further comprising about 1 to about 30% (by weight per volume) of pulverized ECM that has not been solubilized in the hydrogel. (Item 46) The composition according to any one of items 34 to 45, wherein the composition has a storage modulus (G') of about 5 to about 15,000 Pa. (Item 47) The composition is heated at a temperature of approximately 25°C for approximately 0.1 to 1 second. -1 A composition according to any one of items 34 to 46, having a viscosity and a storage modulus of 5 to 15,000 Pa. (Item 48) The composition according to any one of items 34 to 47, wherein the concentration of solubilized ECM in the mammalian acoustic ECM hydrogel is about 20 mg / ml to about 70 mg / ml. (Item 49) The composition according to any one of items 34 to 47, wherein the concentration of solubilized ECM in the mammalian acoustic ECM hydrogel is about 40 to about 66 mg / ml. (Item 50) The composition according to any one of items 34 to 49, wherein the fistula is an anal fistula, and the anal fistula is an intersphincteric fistula, a transsphincteric fistula, a suprasphincteric fistula, an extrasphincteric fistula, or a submucosal fistula. (Item 51) The composition according to any one of items 33 to 49, wherein the composition is for use in the treatment of the fistula by filling the duct of the fistula. (Item 52) The method according to any one of items 1 to 19, or the composition according to any one of items 33 to 51, wherein the fistula is an enterocutaneous fistula (intestine to skin), a cocutaneous fistula (colon to skin), an enteroenteral fistula (intestine to intestine), a vesicocoli fistula (bladder to intestine), a vesicocoli fistula (bladder to colon), a vesicorectal fistula (bladder to rectum), a rectovaginal fistula (vagina to rectum), a vesicovaginal fistula (vagina to bladder), a rectouteritus fistula (uterus to intestine / rectum), a vesicouterine fistula (uterus to bladder), a uretovaginal fistula (ureter to vagina), a retroperitoneal fistula (uterus to abdominal cavity), an enterovaginal fistula (intestine to vagina), a tracheoesophageal fistula, or an anal fistula. (Item 53) The method according to any one of items 1 to 19, or the composition according to any one of items 33 to 51, wherein the fistula is an anorectal, rectovaginal, enteric, tracheoesophageal, biliary-intestinal, bladder-vaginal, bladder-intestinal, pancreatic, cryptoglandular, Crohn's disease, dural venous sinus, colon-bladder, colon-intestinal, colon-vaginal, colon-intestinal, rectourethral, ​​or pharyngeal-cutaneous fistula. (Item 54) The method according to any one of items 1 to 19, or the composition according to any one of items 33 to 53, wherein the fistula is an anal fistula.

Claims

[Claim 1] The invention described in the specification.

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