Acoustic extracellular matrix hydrogels and uses thereof

Abstract

Disclosed herein are methods of making mammalian acoustic extracellular matrix (ECM) hydrogels. In further embodiments, mammalian acoustic ECM hydrogels made using the disclosed methods are disclosed. Also disclosed are mammalian acoustic ECM hydrogels, wherein the hydrogels are thermoreversibly. Methods of using these acoustic ECM hydrogels are also disclosed.

Description

[0001] Cross-references to related applications

[0002] This application is a divisional application of Chinese Patent Application No. 202080020191.1, filed on March 12, 2020, the entire contents of which are incorporated herein by reference.

[0003] This invention claims the benefits of U.S. Provisional Application 62 / 817,787, filed March 13, 2019, and U.S. Provisional Application 62 / 950,565, filed December 19, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to the field of hydrogels, and more particularly to acoustic ECM hydrogels manufactured from mammalian extracellular matrix (ECM) using ultrasound and their uses. Background Technology

[0005] Hydrogels composed of purified ECM components such as collagen, hyaluronic acid, fibroin, laminin, and fibronectin have been widely used in tissue engineering applications. However, these purified single-component ECM biomaterials lack the complex biochemistry of natural tissue ECM. Decellularization of whole tissues or organs provides another method for harvesting ECM that preserves the biochemistry of natural tissue ECM. A major advancement in ECM applications is the ability to form hydrogels, thereby expanding the clinical applicability of ECM. Known techniques for fabricating hydrogels from ECM primarily focus on digesting ECM materials with acidic proteases in acidic solutions; using α-amylase digestion to create ECM foams; or using excision buffers and long dialysis procedures. ECM hydrogels prepared using these techniques inevitably undergo protein degradation and denaturation, thereby diminishing the full complement of ECM molecules and the bioactivity of tissue-specific ECM components. Furthermore, enzyme-based methods for fabricating ECM hydrogels require long incubation times of 24–72 hours to achieve complete dissolution of the ECM components. A method is needed to form ECM hydrogels without using acidic or alkaline solutions and protease digestion. Summary of the Invention

[0006] Disclosed herein are methods for making biocompatible mammalian acoustic extracellular matrix (ECM) hydrogels. These methods include dissolving mammalian ECM in a liquid such as a buffered saline solution at a concentration of 25 milligrams per milliliter to 600 milligrams per milliliter in the liquid with ultrasound at a frequency of about 20 kHz to about 100 kHz at a temperature of 30 to 43 °C for a time sufficient to make an acoustic ECM hydrogel in a liquid phase. In some embodiments, the methods include cooling the acoustic ECM hydrogel in a liquid phase to a temperature of 37 °C or less to make an acoustic ECM hydrogel in a gel phase. In further embodiments, acoustic ECM hydrogels made using the disclosed methods are disclosed.

[0007] Also disclosed are thermoreversibly acoustic ECM hydrogels. In some non-limiting examples, the acoustic ECM hydrogels are in a solid phase at temperatures below about 37 °C and in a liquid phase at temperatures above about 37 °C. These hydrogels are made from mammalian ECM.

[0008] Also disclosed are methods of using these acoustic ECM hydrogels.

[0009] The foregoing and other features and advantages of the present application will become more apparent from the following detailed description of several embodiments, when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figures 1A-1D : Acoustic ECM hydrogels prepared using sonication. (A) Dermal ECM pulverized in a 15 mL conical tube. (B) After resuspending the ECM powder in PBS, the conical tube was placed in an ice water bath and the sonicator probe was inserted into the tube. (C, D) After dissolving the ECM by sonication pulses, the pre-gel solution was pipetted into 3D molds or spread thinly on a Teflon sheet and incubated at temperatures < 37 °C to induce gelation.

[0011] Figures 2A-2D : Representative images of acoustic ECM hydrogels, lyophilized gels, ultrathin ECM sheets, and ECM putty. (A) Acoustic ECM hydrogel cast as a cylinder. (B) Lyophilized acoustic ECM hydrogel retains its 3D morphology. (C) Ultrathin acoustic ECM sheet prepared by casting the ECM gel on a Teflon sheet. (D) ECM putty prepared by sonication of ECM at concentrations below 25 milligrams per milliliter.

[0012] Figure 3 : Scanning electron micrographs (SEM). ECM hydrogels prepared by sonication show textured and fibrous surfaces.

[0013] Figures 4A-4B: Flow sweep. Steady state flow sweep tests were performed on acoustic ECM gels at (A) 25°C, (B) 4 to 37°C and 37 to 4°C. A constant stress was applied to the gel and the resulting deformation was measured. The data show that the viscosity of the gel decreases with greater stress, which is indicative of a shear thinning material.

[0014] Figures 5A-5B : Time sweep test. Time sweep tests were performed on 50 mg / ml acoustic ECM gels at (A) 25°C, (B) 4 to 37°C, and 37 to 4°C to determine the maximum G' (storage modulus) and G" (loss modulus) values. The data show that the storage modulus > loss modulus at all temperatures; that is, it retains the quality of a hydrogel.

[0015] Figures 6A-6C : Typical plot of storage modulus, loss modulus, and complex viscosity of a 50 mg / ml acoustic ECM hydrogel. Data were plotted against angular frequency on a log-log scale measured at 25°C (A), 4°C (B), or by rapidly decreasing the temperature from 37°C to 4°C by applying a small 0.5% oscillatory strain (C). The data show that G' > G" by about an order of magnitude; this indicates that the material meets the criteria of a hydrogel.

[0016] Figure 7 : Flow sweep. Steady state flow sweep tests were performed on acoustic ECM gels at 15°C, 25°C, or 37°C for three different concentrations (25, 100, and 150 mg / ml). The data show that, over a concentration range of 25-150 mg / ml and a temperature range of 15-37°C, at 1, the viscosity of the gel decreases with greater stress, which is indicative of a shear thinning material. Shear thinning refers to the decrease in viscosity with increasing flow (e.g., the "easier" it is to force a material through an opening at the end of a needle or syringe-like device the faster the material is "pushed" through the opening, which is advantageous for clinical applications).

[0017] Figure 8 : Time sweep test. Time sweep tests were performed to determine the maximum G' (storage modulus) and G" (loss modulus) values of acoustic hydrogels at 15°C, 25°C, or 37°C and three different concentrations (25, 100, and 150 mg / ml). The data show that, for all concentrations, at all temperatures, the storage modulus > loss modulus; that is, it retains the quality of a hydrogel.

[0018] Figures 9A-9B : Cell compatibility analysis. (A) 3T3 fibroblasts were seeded onto control (uncoated) or coated with acoustic ECM hydrogels prepared from UBM, SIS, or dermis culture dishes and incubated for 24 hours. Cell viability was assessed using Cell viability was assessed using the MTT Cell Proliferation Assay Kit (Thermo Fisher). Results showed that all ECM cells were non-cytotoxic to 3T3 fibroblasts (n=3). (B) Live / Dead Analysis. Horse mesenchymal stem cells were seeded onto hydrogel-coated plates and compared to cells grown on tissue culture plastic. Viability was assessed using a live / dead analysis kit (Invitrogen). Five 200X images were captured in three technical replicates. The percentage of live and dead cells was quantified using Cell Profiler. Error bars represent standard deviation.

[0019] Figure 10 The Lee White coagulation method was used to determine the hemostatic powder AVITENE prepared into hydrogels by acoustic methods. TM and XENMATRIX TM ECM coagulation time (hemostasis). XENMATRIX TM It is an ECM product collected from pig dermis. Data shows that, compared to untreated AVITENE... TM and XENMATRIX TM The gel achieves rapid hemostasis.

[0020] Figure 11 In vivo evaluation of coagulation time using a rat liver rupture model. Rats underwent liver rupture and were treated with hemostatic agents. Sprague-Dawley rats were randomly assigned to 5 experimental groups (n=5 per group): Arista powder (BD / CRBard), AVITENE... TM Powder (BD / CR Bard), Micromatrix powder (ACell), esophageal ECM prepared into hydrogels using acoustic methods at indicated concentrations, and XenMatrix (BD / CR Bard) prepared into hydrogels using acoustic methods at indicated concentrations. Data show that mammalian ECM prepared into hydrogels using acoustic methods can induce hemostasis in vivo.

[0021] Figures 12A-12C Acoustic hydrogels were prepared by acoustic treatment of the ECM at a frequency of 20 kHz using amplitudes ranging from 20-100%. Samples were all 50 mg / mL and acoustically treated for 10 minutes before experiments were conducted at 15 °C. (A) Image of the hydrogel formed at the indicated amplitudes. (B) Flow viscosity. Rheological data show that, at all tested amplitudes, the viscosity of the gel decreases with increasing stress, suggesting a shear-thinning material. (C) Time scan. Rheological data show that, for all concentrations and at all amplitudes, the storage modulus > loss modulus; that is, it maintains the quality of the hydrogel.

[0022] Figure 13 UBM acoustic hydrogel promotes M2-like macrophage phenotype. Mouse bone marrow-derived macrophages were treated with 2 mg / ml UBM acoustic hydrogel for 24 hours, fixed and immunolabeled for pro-inflammatory M1-like markers (iNos, TNFa) or strong indicators of pre-remodeled M2-like markers (Izz1, Arginase) and counterstained with DAPI. Cells treated with IFNg and lipopolysaccharide (LPS) were used as positive controls for M1-like phenotype, and IL-4 was used as positive control for M2-like phenotype. F4 / 80 staining was used as positive control for macrophages. Cells were imaged at 200X. Data show that UBM acoustic hydrogel promotes M2-like macrophage phenotype compared to controls.

[0023] Figures 14A-14F Sonoporation and temperature-induced gelation of comminuted ECM. (A) Demonstration of sonicator tip immersion depth in 50 ml conical tube. (B, C) Comminuted skin ECM powder in IX PBS before (B) and after (C) sonication. (D) Inversion of tube shows that solubilized ECM polymerizes into a hard gel after incubation at temperatures below 25 °C. (E) Polymerized gel can conform to 3D geometry. (F) Solubilized ECM can be transferred to a syringe (top panel), which can then be cooled to temperatures below 25 °C to produce a gel in injectable form (bottom panel).

[0024] Figures 15A-15D Solubilization of collagen and sulfated glycosaminoglycans (sGAGs). (A) Concentration of solubilized collagen as a function of sonication amplitude. Comminuted dECM was sonicated for 300 seconds at the indicated amplitudes. SIRCOL TM Analysis measured the concentration of solubilized collagen. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscripts indicate pair-wise comparisons. (B) Concentration of sGAGs as a function of sonication amplitude. Comminuted dECM was sonicated for 300 seconds at the indicated amplitudes. BLYSCAN TM Analysis measured the concentration of solubilized sGAGs. Data are presented as mean ± s.d. for n = 3 samples per group. (C) Concentration of solubilized collagen as a function of sonication time. Comminuted dECM was sonicated at 100% amplitude for the indicated times. SIRCOL TM Analysis measured the concentration of solubilized collagen. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscripts indicate pair-wise comparisons. (D) Concentration of solubilized sGAGs as a function of sonication time. Comminuted dECM was sonicated at 100% amplitude for the indicated times. BLYSCAN TMThe concentration of solubilized sGAG was analyzed. Data are presented as mean ± s.d. for n = 3 samples per group.

[0025] Figures 16A-16C Effect of temperature and sonication amplitude on gelation time of ECM hydrogels prepared using ultrasonic cavitation. (A) Effect of temperature on gelation time. 25, 50, and 100 mg / ml dECM were sonicated at 100% amplitude for 300 s and then incubated at the indicated temperatures to induce gelation. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscripts indicate pair-wise comparisons. (B) Effect of sonication amplitude on gelation time. 25, 50, and 100 mg / ml dECM were sonicated at the indicated amplitudes for 300 s and then incubated at 4°C to induce gelation. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscripts indicate pair-wise comparisons. (C) Gelation analysis to evaluate the effect of temperature on gelation time of UBM, SIS, eECM, tECM, or LECM. 100 mg / ml concentration of the indicated tissue ECMs were sonicated at 100% amplitude for 300 s and then incubated at 4°C or 25°C to induce gelation. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscripts indicate pair-wise comparisons.

[0026] Figures 17A-17C Viscoelastic characterization of ECM hydrogels prepared using ultrasonic cavitation. (A) Typical plot of ECM hydrogel gelation kinetics of dECM and eECM under 3 temperature profiles. The storage modulus (G') S-shaped increase when temperature is rapidly decreased (37→4°C). The hydrogel stiffness (G') is maintained when temperature is rapidly increased (4→37°C and 25→37°C). (B) Average storage modulus under 3 temperature profiles (n = 3, mean ± SD). (C) Average time to reach 50% gelation for the S-shaped temperature profile 37→4°C (n = 3, mean ± SD). * p < 0.05, ** p < 0.01.

[0027] Figures 18A-18E In vitro cellular response. (A) 3T3 fibroblasts were seeded on control (uncoated) or coated with ECM hydrogels prepared from UBM, SIS, or dermis and cultured for 24 h. Cell proliferation was assessed using MTT cell proliferation assay kit to evaluate cell metabolic activity. Data presented as mean ± s.d. for n=3 samples per group. (B, C) Live / dead assay. Primary equine mesenchymal stem cells were plated on control (uncoated) or culture dishes coated with ECM hydrogels prepared from dECM or UBM. Viability was evaluated using a live / dead assay kit. Cells were imaged at 200X (B) and the percentage of live and dead cells was quantified using Cell Profiler (C). Data presented as mean ± s.d. for n=3 samples per group. (D) Murine bone marrow-derived macrophages were untreated (control) or treated for 24 hours with the following test articles: IFNy+LPS, IL-4, dECM hydrogel, or eECM hydrogel. Cells were immunolabeled with F4 / 80 (macrophage marker), iNOS (Ml-like marker), or Fizzl (M2-like marker). Cells were imaged at 200X. (E) Quantification of F4 / 80, iNOS, and Fizzl immunolabeling. Data presented as mean ± s.d. for n=3 per group.

[0028] Figures 19A-19C Acoustic hydrogels as submucosal fluid bolus. (A) Acoustic extracellular matrix (ECM) hydrogels (100 mg / ml) were prepared from dermal ECM (dECM) and esophageal mucosal ECM (eECM) and used as submucosal fluid bolus in vitro. The effect of 20 kGy gamma irradiation (y) on the height of the acoustic ECM hydrogel fluid bolus was evaluated. Clinical standard Eleview and PBS were used for controls. Fluid bolus height was measured after injection of 2 ml of test article in the pig esophagus over time. Values expressed as mean + / - SD (n=3). (B) Acoustic hydrogel samples were injectable through a 16G syringe. (C) Typical picture of test article fluid bolus height after 75 minutes.

[0029] Figures 20A-20B Acoustic hydrogels gelation. The hydrogel "stiffness" of gamma irradiated (20 kGy) and unsterilized control acoustic hydrogels (dermal ECM 100 mg / ml) was measured over time. The storage modulus ("stiffness") (G') and loss modulus (G") were measured by applying a small 0.5% oscillatory strain to the sample. Three temperature profiles were tested: the temperature was rapidly increased from an initial storage temperature to a final temperature: 4 to 37°C, 25 to 37°C, or 37 to 4°C. (A) Typical plot showing the time scan. (B) Average storage and loss modulus averaged over the last 5 minutes of the test are shown. DETAILED DESCRIPTION

[0030] ECM hydrogels have been used as substrates for 3D organoid culture and for promoting repair and reconstruction of a variety of tissues in many preclinical and clinical applications. Previous ECM hydrogel materials were fabricated using lengthy methods that focused on enzymatic digestion of ECM with acid proteases in acidic solutions; or used chaotropic extraction buffers and dialysis procedures that can affect the structure and function of native proteins. Disclosed herein is a method for preparing hydrogels from ECM bioscaffolds using ultrasonic cavitation. By adjusting the temperature, the solubilized ECM can be induced to rapidly self-assemble into a gel, and by adjusting the ECM concentration and sonication parameters, the material properties of the gel can be tailored. ECM bioscaffolds can be successfully solubilized using ultrasound without the need for enzymatic digestion and induced to re-polymerize into a gel form that is capable of supporting cell growth. These hydrogels can be used in a variety of applications and can ultimately be terminally sterilized with gamma irradiation.

[0031] For the manufacture of the disclosed ECM hydrogels, the sonication technique can be applied to a wide range of tissue-specific ECMs, including but not limited to dermis, urinary bladder matrix (UBM), and small intestinal submucosa (SIS). It can also be used with commercially available ECM preparations. In some embodiments, the method includes resuspending a pulverized ECM in a liquid, such as a buffer, such as a neutral buffered saline solution, and then solubilizing the ECM with ultrasound. In some embodiments, the buffered saline solution has an osmolarity of about 290 mOsm / L. Various concentrations can be used, and the ECM can be sonicated for at least 60 seconds. Rapid gelation of the ECM solution can be induced by lowering the temperature of the ECM solution. The gelation time and ECM gel properties can be adjusted by adjusting the ECM concentration, sonication amplitude, and time. In some embodiments, the acoustic ECM hydrogels do not contain exogenous proteases or inactivated exogenous proteases, such as exogenous pepsin, trypsin, or hyaluronidase, or inactivated forms of exogenous pepsin, trypsin, or hyaluronidase.

[0032] Once polymerized, these ECM hydrogels are stable at room temperature and can conform to customizable 3D geometries. The ECM hydrogels fabricated by sonication ("acoustic ECM hydrogels") can be processed into solid scaffolds that maintain the overall 3D geometry and increase porosity through freezing and lyophilization procedures. This technique can support the incorporation of cells or compounds for in vitro and in vivo applications. Methods of using the disclosed acoustic ECM hydrogels are also disclosed, such as but not limited to increasing hemostasis.

[0033] There has been relatively little progress in large-scale production of ECM hydrogels (Brown et al., supra, 2012). The disclosed methods are used for large-scale production in several ways. In some embodiments, the concentration range of ECM hydrogels can be extended from 2-20 milligrams / milliliter (limit of enzymatic methods) to 25-100 milligrams / milliliter using ultrasonic cavitation methods, which allows for fine-tuning of the ECM hydrogel viscoelastic properties for specific clinical applications. In other embodiments, the processing time is significantly reduced from 48-72 hours to the order of minutes. In further embodiments, the ECM hydrogels can conform to customizable 3D geometries and can support the incorporation of cells or therapeutic drugs for in vitro and in vivo applications.

[0034] Terminology

[0035] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of commonly used 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, 16 volumes, 2008; and other similar references.

[0036] To facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0037] Acid protease: an enzyme that cleaves peptide bonds, wherein the enzyme has increased activity in cleaving peptide bonds at acidic pH. For example and without limitation, acid proteases can include pepsin and trypsin.

[0038] Antibiotic: a compound or substance that kills or significantly slows the growth of bacteria, fungi, or any other microorganism. An "antibacterial agent" is a compound or substance that kills or significantly slows the growth of bacteria.

[0039] Antibacterial antibiotics are generally classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target a bacterial function or growth process. Those that target the bacterial cell wall (e.g., penicillins and cephalosporins) or cell membrane (e.g., polymyxins) or 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. Additional classifications are based on their target specificity.

[0040] "narrow-spectrum" antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria. "Broad-spectrum antibiotics" affect many different types of bacteria. Antimicrobials also include cyclic lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), and oxazolidinones (e.g., linezolid).

[0041] Topical antibiotics are antibiotics that are applied to the surface of the body, such as the skin or the eye. Topical antibiotics are usually formulated as ointments or creams and contain active agents such as macrolide antibiotics (e.g., erythromycin), sulfonamide antibiotics (e.g., sulfacetamide), cyclic peptides (e.g., bacitracin, polymyxin), psuedomonic acid (e.g., mupirocin), aminoglycosides (e.g., neomycin), or quinolones (e.g., ciprofloxacin or ofloxacin), nitroimidazoles (e.g., metronidazloe), or drug combinations (e.g., bacitracine / polymyxin or neomycin / polymyxin B / bacitracin).

[0042] Biocompatible: Any material that does not cause an adverse response in a subject when implanted in a mammalian subject. A biocompatible material, when introduced into an individual, is capable of performing its intended function and is not toxic or injurious to that individual nor does it induce immune rejection of the material in the subject.

[0043] Biological scaffold: A biocompatible scaffold, usually a solid support or a gel. A biological scaffold is composed of naturally occurring materials. A "biosynthetic scaffold" is composed of non-naturally occurring and naturally occurring materials.

[0044] Centrifugation: This method involves the application of a centrifugal force to a mixture, whereby the more dense components in the mixture migrate away from the axis of the centrifuge relative to the other less dense components in the mixture. The force applied to the mixture is a function of the speed of the centrifuge rotor and the radius of rotation. In most applications, the rotational force will cause a pellet (small particle) to collect at the bottom of the centrifuge tube, with the remaining solution appropriately termed the "supernatant" or "upper layer". In other similar applications, density-based separation or "gradient centrifugation" techniques are used to isolate specific materials from mixtures containing both more dense and less dense components than the desired component.

[0045] During the circular motion of the centrifuge rotor, the force applied is the product of the radius of rotation and the angular velocity, where the force is traditionally expressed as an acceleration relative to "g", the standard gravitational acceleration at the surface of the Earth. The applied centrifugal force is referred to as the "relative centrifugal force" (RCF) and is expressed in multiples of "g".

[0046] Pulverization: The process of reducing larger particles to smaller particles, including but not limited to by grinding, blending, mincing, slicing, milling, cutting, shredding. ECMs can be pulverized, although in any form, including but not limited to, hydrated form, frozen, air-dried, lyophilized, powder, tablet form.

[0047] Contact: Placed in a manner that is directly physically joined, which can be in solid or liquid form.

[0048] Cytokine: The term "cytokine" is used as a generic name for a diverse group of soluble proteins and peptides that act at nanomolar to picomolar concentrations as body fluid mediators and modulate the functional activities of individual cells and tissues under normal or pathological conditions. These proteins also directly mediate cell-to-cell interactions and regulate processes that occur in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor-alpha, interleukin (IL)-6, IL-10, IL-12, transforming growth factor, and interferon-gamma.

[0049] Diagnosis: The process of identifying a disease through its signs, symptoms, and the results of various tests. The conclusion reached through this process is also referred to as a "diagnosis." Forms of testing commonly performed include blood tests, medical imaging, and biopsies.

[0050] Extracellular Matrix (ECM): A natural acellular scaffold for cell growth. Natural ECM (ECM found in multicellular organisms, such as but not limited to mammals and humans) is a complex mixture of structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. In mammals, ECM typically comprises about 90% collagen in its various forms. The composition and structure of ECM varies depending on the tissue source. For example, small intestinal submucosa (SIS), urinary bladder matrix (UBM), esophageal (E), and liver matrix ECMs each differ in their overall structure and composition due to the unique cellular niches required for each tissue. An "intact extracellular matrix" and "intact ECM" is an extracellular matrix that retains the activity of its structural and non-structural biomolecules, including but not limited to collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0051] The structure and / or activity of biomolecules in the ECM can be chemically or mechanically altered or removed, for example, by crosslinking and / or by dialysis of the ECM. An "intact" ECM is essentially free of enzymatic digestion, crosslinking, and / or dialysis, meaning that the ECM has not been subjected to a digestion, dialysis, and / or crosslinking process prior to solubilization, or conditions other than those that occur naturally during storage and handling of the ECM. Thus, an ECM that is significantly crosslinked and / or dialyzed (in any but a trivial way that does not significantly affect the gelling and functional properties of the ECM in its uses described herein) is not considered "intact." "Cell-free" refers to an ECM manufactured from a tissue of origin that has been treated to remove cells, leaving the ECM. Decellularized tissue is used to manufacture ECM hydrogels.

[0052] Gel: A state of matter between liquid and solid, and is generally defined as a crosslinked polymer network swollen in a liquid medium. Generally, a gel is a two-phase colloidal dispersion containing both solid and liquid, where the amount of solid is greater than in a two-phase colloidal dispersion called a "sol." As such, a "gel" has some properties of a liquid (i.e., shape is elastic and deformable) and some properties of a solid (e.g., shape is sufficiently discrete to maintain three dimensions on a two-dimensional surface). "Gel time," also called "gel time," refers to the time it takes for a composition to become non-flowable under moderate stress.

[0053] Gelation: Formation of a gel from a sol.

[0054] Hemostasis: The inhibition or cessation of bleeding.

[0055] Hydrogel: A network of hydrophilic polymer chains, sometimes found in the form of a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also have a degree of flexibility similar to natural tissue. "Acoustic" hydrogels, such as acoustic ECM hydrogels, are manufactured using ultrasonic energy. The characteristics of these hydrogels are disclosed herein. For hydrogels, G' (storage modulus) is typically greater than G" (loss modulus) by about an order of magnitude.

[0056] Isolated: A biological component (e.g., extracellular matrix) that has been substantially isolated, manufactured apart from, or purified apart from other biological components, cells, or organisms in which the component naturally occurs, i.e., living cells, other chromosomes and extrachromosomal DNA and RNA, and proteins. Thus, nucleic acids, peptides, and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An isolated ECM has been separated from the cells that manufactured the ECM.

[0057] Isotonic buffer solution: a solution that is buffered to a pH of 7.2-7.8 and has a balanced concentration of salts to promote an isotonic environment.

[0058] Macrophage: a type of white blood cell that engulfs and degrades cellular debris, foreign substances, microorganisms, and cancer cells. In addition to their role in phagocytosis, these cells play important roles in development, tissue maintenance and repair, and innate and adaptive immunity, as they recruit and influence other cells, including immune cells such as lymphocytes. Macrophages can exist in a number of phenotypes, including those known as Ml and M2, also known as "Ml -like" and "M2-like." Macrophages that perform primarily pro-inflammatory functions are known as Ml macrophages (CD86+ / CD68+), while macrophages that reduce inflammation and promote and regulate tissue repair are known as M2 macrophages (CD206+ / CD68+). Markers that identify various phenotypes of macrophages are different between species. It should be noted that macrophage phenotypes are represented by a spectrum ranging between Ml and M2 extremes.

[0059] Mammal: this term includes humans and non-human mammals. Similarly, the term "subject" includes human and veterinary subjects.

[0060] Preventing or treating a disease: "preventing" a disease refers to inhibiting, either partially or completely, development of a disease, for example, in a person known to be susceptible to the disease, such as cancer. An example of a person known to be susceptible is someone with a family history of breast cancer, or someone who has been exposed to a factor that makes the subject susceptible to a condition, such as melanoma. "Treating" refers to therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. In some embodiments, treatment refers to reduction in tumor size, reduction in the number and / or size of metastases, or reduction in symptoms of a tumor.

[0061] Therapeutic agent: used in a general sense, it includes therapeutic, prophylactic, and replacement agents. "Treatment" or "treating" refers to providing a patient with a substance, such as an acoustic ECM hydrogel, in an amount sufficient to measurably affect a biological parameter, such as increasing hemostasis.

[0062] Therapeutically effective amount: A "therapeutically effective amount" of a composition, such as an acoustic ECM hydrogel, is an amount effective, when administered to a patient, to provide a therapeutic benefit, such as an improvement of symptoms, a reduction in progression, or an induction of disease regression. The amount of acoustic ECM hydrogel is sufficient to achieve the intended effect in the subject being treated. A therapeutically effective amount can be administered systemically or locally, for example to a wound. Furthermore, an effective amount of acoustic ECM hydrogel can be administered in a single dose or in several doses over time. However, the effective amount will depend on the formulation administered, the subject being treated, the severity and type of affliction, and the manner of administration of the compound. Acoustic ECM hydrogels for use in the methods disclosed herein have equal application in medical and veterinary settings. Thus, the general term "subject" or "patient" is understood to include all animals, including but not limited to human or veterinary subjects, such as other primates, dogs, cats, horses, and cows.

[0063] Thermoreversible hydrogel: A hydrogel formed due to entanglement of polymer chains, where the viscosity changes at the characteristic temperature of gelation. The disclosed acoustic ECM hydrogels are thermoreversible hydrogels, which show gelation (sol to gel transition) upon cooling.

[0064] Topical administration: A medicament that is administered topically is applied only to a specific area, rather than throughout the body. In a particular example, a composition is applied to the skin or eye in an area where hemostasis is desired. For example, a pharmaceutical composition can be administered in the form of a topical formulation to a wound, such as an epithelial wound or defect, such as a traumatic or surgical wound, such as a skin or corneal abrasion or a surgical incision.

[0065] Ultrasonication: A process of exposing ultrasonic waves at a frequency higher than 20 kHz.

[0066] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given are approximate, and are provided for description. "About" indicates that a value is within 5% of the recited value. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification will control. In addition, the section headings included herein are for organizational purposes only and are not meant to be used in construing the terms of the disclosure. Further, materials, methods, and examples are illustrative only and not intended to be limiting.

[0067] Extracellular matrix (ECM)

[0068] Any type of extracellular matrix tissue can be used to make the hydrogel (see U.S. Patents 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; 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 relating to ECM). In certain embodiments, the ECM is isolated from a vertebrate, such as and without limitation, a warm-blooded mammalian vertebrate, including but not limited to, humans, monkeys, horses, pigs, cows, and sheep. In a specific, non-limiting example, the ECM is porcine or human.

[0069] The ECM can be derived from any organ or tissue, including but not limited to bladder, intestine (e.g., small or large intestine), heart, kidney, uterus, brain, blood vessel, lung, skeletal muscle, pancreas, stomach, spleen, adipose tissue, liver, esophagus, and dermis. The ECM can be obtained from cell culture. In one embodiment, the ECM is isolated from bladder. In another embodiment, the ECM is from esophagus. In another embodiment, the ECM is from dermis. The ECM can or can not include a basement membrane portion of the ECM. In certain embodiments, the ECM includes at least a portion of the basement membrane. The tissue can be decellularized to remove cells and cellular material from, for example, the source tissue or organ, to manufacture the ECM. It is desirable to use decellularized material to prevent an immune response, for example, when the ECM is implanted into a subject, for example, as a component of a hydrogel disclosed herein. Removal of cellular material, for example, when using the ECM to form a hydrogel, prevents such an immune response.

[0070] U.S. Patent 8,361,503 (incorporated herein by reference) discloses preparation of bladder ECM, for example, porcine bladder ECM, by rubbing the bladder tissue to remove the outer layers including the serosa and muscularis using a longitudinal rubbing motion with a scalpel handle and moistened gauze. After evertion of the tissue segment, the luminal portion of the mucosa is peeled from the underlying tissue using the same rubbing motion. In some embodiments, the submucosa is prevented from perforating. After removal of these tissues, the resulting ECM consists primarily of the submucosa.

[0071] Manufacture of hydrogels from dermal ECM is disclosed in Wolf et al., Biomaterials 33:7028-7038, 2012, incorporated herein by reference. Manufacture of ECM from esophageal tissue is disclosed, for example, in Badylak et al., J Pediatr Surg. 35(7): 1097-103, 2000 and Badylak et al., J Surg Res. 2005 Sep; 128(1): 87-97, 2005, both incorporated herein by reference. U.S. Patent 6,893,666, incorporated herein by reference, discloses manufacture of ECM from bladder, skin, esophagus, and small intestine. ECM can be manufactured from any of these tissues.

[0072] Commercially available ECM preparations can also be used. In one embodiment, the ECM is derived from small intestinal submucosa or SIS. Commercially available preparations include, but are not limited to, SURGISIS® TM , SURGISIS-ES TM , STRATASIS TM , and STRATASIS-ES TM (Cook Urological Inc.; Indianapolis, Ind.) and GRAFTPATCH®TM (Organogenesis Inc.; Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available formulations include, but are not limited to, PELVICOL. TM (in Europe, PERMACOL) TM Sales; Bard, Covington, Ga.), REPLIFORM TM (Microvasive; Boston, Mass.) and ALLODERM TM (LifeCell; Branchburg, NJ). In another embodiment, the ECM is derived from the bladder. Commercially available formulations include, but are not limited to, UBM (Acell Corporation; Jessup, Md.).

[0073] Tissues used for preparing ECM can be collected in a variety of ways, and once collected, multiple portions of the collected tissue can be used. ECM has also been prepared from the esophagus and small intestine; see, for example, Keane et al., Tissue Eng. Part A, 21(17-18):2293-2300, 2015, incorporated herein by reference. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the outer muscle layer, digesting the mucosal layer in a buffer containing trypsin, and then exposing it to sucrose. Deoxycholic acid, peracetic acid, and DNAse are used to prepare the submucosal layer of the small intestine (SIS). SIS can be prepared by mechanically removing the superficial layers of the mucosa, serosa, and outer muscularis propria from the intact small intestine, preserving the integrity of the submucosal layer, muscularis mucosae, and basal compacta. The SIS is then treated with peracetic acid. Keane et al. provide an exemplary protocol. For example, skin hydrogels can be prepared 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.

[0074] In one embodiment, the ECM is isolated from a collected pig bladder to prepare bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. The serosa, outer muscular layer, submucosa, and most of the muscular mucosa can be removed by mechanical abrasion or by a combination of enzymatic treatment, hydration, and abrasion. Mechanical removal of these tissues can be accomplished by abrasion using longitudinal wiping motions to remove the outer layers (particularly the abluminal smooth muscle layer) and even the luminal portion of the mucosa (epithelial layer). Mechanical removal of these tissues is accomplished by removing mesenteric tissue with, for example, Adson-Brown forceps and Metzenbaum scissors, and wiping away the muscular layer and submucosa with a longitudinal wiping motion using a scalpel handle or other rigid object wrapped in moist gauze. The epithelial cells of the mucosa can also be dissociated by immersing the tissue in an ablation solution, such as, but not limited to, hypertonic saline. The resulting UBM comprises a base membrane of mucosa and adjacent intrinsic membrane, which is further treated with peracetic acid, lyophilized and powdered, see U.S. Patent 8,361,503, which is incorporated herein by reference.

[0075] The dermal fraction can be used to prepare ECM hydrogels, see PCT application 2015 / 15164728, which is incorporated herein by reference. In a specific, non-limiting example, the dermal fraction can be prepared with 0.25% trypsin / 1%... (i.e., without SDS) Decellularize at 300 RPM at room temperature on a vortex mixer in the following solutions: 0.25% trypsin, 6 h, lx; deionized water, 15 min, 3x; 70% ethanol, 10 to 12 h, lx; 3% H2O2, 15 min, lx; deionized water, 15 min, 2x; 1% H2O2. / 0.26% EDTA / 0.69% Tris, 6 h, lx and then overnight, lx; deionized water, 15 min, 3x; 0.1% peracetic acid / 4% ethanol, 2 h, lx; PBS, 15 min, 2x; and finally deionized water, 15 min, 2x. The leather sheets were then lyophilized and subsequently shrunk into granules using a Waring mixer and a Wiley mill with a #20 mesh sieve.

[0076] In some embodiments, the epithelial cells can be first delaminated by first soaking the tissue in a de-epithelializing solution, such as a hypertonic saline, for example and without limitation, 1.0 N saline, for a period of time ranging from 10 minutes to 4 hours. Exposure to the hypertonic saline solution effectively removes the epithelial cells from the underlying basement membrane. The tissue remaining after the initial delamination procedure includes the epithelial basement membrane and the tissue layers that are abluminal to the epithelial basement membrane. This tissue is then subjected to further processing to remove most of the abluminal tissue but not the epithelial basement membrane. The outer serosa, tunica externa, smooth muscle tissue, submucosa, and most of the muscularis mucosa are removed from the remaining de-epithelialized tissue by mechanical abrasion or by a combination of enzymatic treatment, hydration, and abrasion.

[0077] The ECM can be sterilized by a 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 obtained by soaking in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water for two hours. 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 treatment (0.05-4 mRad), gas plasma sterilization, peracetic acid sterilization, or electron beam treatment. The ECM can also be sterilized by glutaraldehyde treatment, which induces cross-linking of the protein material, but this treatment significantly alters the material such that it is slowly resorbed or not resorbed at all and induces a different type of host remodeling that more closely resembles scar tissue formation or encapsulation rather than constructive remodeling. Cross-linking of the protein material can also be induced with carbodiimides or dehydrothermal or photo-oxidative methods. As disclosed in U.S. Patent 8,361,503, the ECM is sterilized by immersion in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 96% (v / v) sterile water for 2 hours. The ECM material is then washed twice for 15 minutes with PBS (pH = 7.4) and twice for 15 minutes with deionized water.

[0078] Generally, after the tissue under consideration is isolated, decellularization is performed by various methods, such as and without limitation, exposure to hypertonic saline, peracetic acid, or other detergent. Sterilization and decellularization can be performed simultaneously. For example and without limitation, the above sterilization with acetic acid can also be used for decellularization. The ECM can then be dried, lyophilized (freeze-dried), or air-dried. The dried ECM can be comminuted by methods including, but not limited to, tearing, grinding, cutting, milling, and shearing. The comminuted ECM can also be further processed into a powdered form by methods such as, and without limitation, grinding or milling in a frozen or freeze-dried state.

[0079] Mammalian ECMs are also commercially available. These include AVITENE TM , XENMATRIX TM , and XENMATRIX TM .

[0080] Acoustic ECM hydrogels and preparation thereof

[0081] In some embodiments, the pulverized ECM, such as a mammalian ECM, is diluted in a liquid. The ECM can or can not be lyophilized prior to pulverization. The ECM can be pulverized by, for example, grinding, shredding, or cutting the ECM. The pulverized ECM should have pieces in the range of about 10 μιη to about 5000 μιη, about 10 μιη to about 4000 μιη, about 10 μιη to about 3000 μιη, about 10 μιη to about 2000 μιη, about 10 μιη to about 1000 μιη, about 10 μιη to about 500 μιη, about 30 μιη to about 300 μιη, about 40 to about 400 μιη, about 25 μιη to about 500 μιη, about 50 μιη to about 500 μιη, about 100 μιη to about 300 μιη, about 10 μιη to about 50 μιη, or about 10 μιη to about 100 μιη. In one embodiment, the ECM is provided in the form of pieces in the range of about 10 μιη to about 1000 μιη. In another preferred embodiment, the ECM is provided in the form of pieces in the range of about 10 μιη to about 2000 μιη. In one non-limiting example, the pieces are in the range of about 30 μιη to about 300 μιη. The liquid can be a neutral pH buffer, for example, a pH of about 7.0 to about 7.6, such as about 7.1 to about 7.5, such as about 7.2 to about 7.4, such as about 7.0 to 7.2, such as about 7.0 to 7.4, such as about 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. The ECM can be diluted in an isotonic buffered saline solution, such as, but not limited to, phosphate buffered saline (PBS) or Tris buffered saline. In some embodiments, the buffered saline solution has an osmolarity of about 290 mOsm / L. The liquid can be water. In some embodiments, isotonic buffers, including but not limited to phosphate buffered saline (PBS), can be used to bring the solution to the target pH, or to help maintain the pH and ionic strength of the gel at target levels, such as physiological pH and ionic conditions. This forms a liquid ECM solution.

[0082] The disclosed methods generally do not involve the use of acid proteases, including pepsin, trypsin, or hyaluronidase. See PCT Application WO 2015 / 164728, incorporated herein by reference. Generally, in the present methods, the ECM dissolved in the liquid is not contacted with acid proteases.

[0083] In some embodiments, the ECM is used in a liquid at a concentration greater than about 25 mg / ml. The ECM can be used in a liquid, such as a buffer, at a concentration of about 25 mg / ml to about 600 mg / ml. Suitable concentrations also include 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. The ECM can be used in a liquid, such as a buffer, at a concentration of about 50 mg / ml to about 600 mg / ml. Suitable concentrations also include about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml, and about 50 mg / ml to about 150 mg / ml. Suitable concentrations include about 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 about 25 mg / ml, 100 mg / ml, and 150 mg / ml. In one non-limiting example, the ECM is at a concentration of about 25 mg / ml to about 150 mg / ml in a liquid. In one non-limiting example, the ECM is at a concentration of 100 mg / ml in a liquid.

[0084] The ECM in a liquid, such as a buffered saline solution, is treated with ultrasound frequencies. In one embodiment, the ultrasound is at a frequency of about 20 kHz to about 100 kHz. The ECM in a liquid can be treated with ultrasound at a frequency of about 20 kHz to about 30 kHz, about 20 kHz to about 40 kHz, about 20 kHz to about 50 kHz, about 20 kHz to about 60 kHz, about 20 kHz to about 70 kHz, about 20 kHz to about 80 kHz, or about 20 kHz to about 90 kHz. The ECM in a liquid can be treated with ultrasound at a frequency of about 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, or 100 kHz. In one non-limiting example, the ECM in a liquid can be treated with ultrasound at a frequency of about 20 kHz.

[0085] The ECM in the liquid, such as a buffered saline solution, is treated with ultrasound for at least 20 seconds, such as at least 30 seconds. The ECM in the liquid, such as a buffered saline solution, is treated with ultrasound for at least 60 seconds. In some embodiments, the ECM in the liquid is treated with ultrasound for at least 60 seconds to about one hour. In further embodiments, the ECM in the liquid is treated with ultrasound for at least 60 seconds to about 30 minutes. In further embodiments, the ECM in the liquid is treated with ultrasound for at least 30 seconds to about 30 minutes. In further embodiments, the ECM in the liquid is treated with ultrasound for at least 60 seconds to about 15 minutes. In further embodiments, the ECM in the liquid is treated with ultrasound for at least 30 seconds to about 15 minutes. In some embodiments, the ECM in the liquid is treated with ultrasound for at least 60 seconds to about 10 minutes. In some embodiments, the ECM in the liquid is treated with ultrasound for at least 30 seconds to about 10 minutes. In some embodiments, the ECM in the liquid is treated with ultrasound for at least 60 seconds to about 5 minutes. In some embodiments, the ECM in the liquid is treated with ultrasound for at least 30 seconds to about 5 minutes. The ECM in the liquid can be treated with ultrasound for about 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, the ECM in the liquid is treated with ultrasound in a pulsed form for the total time listed herein. Thus, in some embodiments, the ECM in the liquid, such as a buffered saline solution, is treated with pulses, the length of which is, for example, at least about 30 seconds, such as about 30, about 40, or about 60 seconds. The ECM in the liquid, such as a buffered saline solution, can be treated with ultrasound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, such that the total treatment time is 60 seconds to one hour, or any of the total times listed. The ECM in the liquid, such as a saline solution, 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. The ECM in the liquid, such as a saline solution, can be treated for at least 30 seconds. Typically, if multiple treatments are used, they occur within less than 1 hour. An exemplary method is a 30 second pulse of ultrasound, followed by 30 to 45 seconds of no treatment, followed by another treatment. This treatment is applied 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more.One exemplary non-limiting method is six pulses of ultrasound for 30 seconds, e.g., about 20 kHz, followed by 45 seconds off, repeated six times, for a total of 3 minutes of sonication.

[0086] The ultrasound can have an amplitude of about 20 pm to about 320 pm. Typically, the amplitude is measured from the center of the probe used to generate the ultrasound. The amplitude of the probe vibrating surface is the distance between its position in the fully extended and fully retracted state, measured in micrometers (pm). In some embodiments, the amplitude is about 30 pm to about 200 pm. In further embodiments, the amplitude is about 36 pm to about 180 pm. The amplitude can be about 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 pm. In some embodiments, the amplitude can be about 30-40 pm, 40-50 pm, 50-60 pm, 60-70 pm, 70-80 pm, 80-90 pm, 90-100 pm, 100-110, 110-120 pm, 120-130 pm, 130-140 pm, 140-150 pm, 150-160 pm, 160-170 pm, 170-180 pm, 180-190 pm, 190-200 pm, 200-210 pm, 210-220 pm, 220-230 pm, 230-240 pm, 240-250 pm, 250-260 pm, 260-270 pm, 270-280 pm, 280-290 pm, or 290-300 pm. In one specific non-limiting example, the frequency of the ultrasound is about 20 kHz, and the amplitude is about 36 pm to about 180 pm. In another non-limiting example, the frequency of the ultrasound is about 20 kHz, and the amplitude is about 36 pm to about 180 pm, and the treatment is for a total of about 1, 2, 3, 4, or 5 minutes, such as about 3 minutes. The sonication can be for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. The sonication can be for about 30 seconds to about 5 minutes. The sonication can be, for example, for about 1 to about 5 minutes. The sonication can be for about 1 to about 10 minutes. The sonication can be, for example, for 1 to about 20 minutes. In further embodiments, the sonication can be for less than about 1 hour, less than about 30 minutes, less than about 20 minutes, or less than about 10 minutes. In some embodiments, the sonication can be for at least 30 seconds. In other embodiments, the sonication can be for 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 sonication can be for up to 48 hours.

[0087] In some embodiments, the ECM in a liquid is treated with ultrasound at a temperature ranging from about 30 °C to about 43 °C. In one embodiment, the ECM in a liquid is treated with ultrasound at a temperature ranging from about 35 °C to about 40 °C. In one embodiment, the ECM in a liquid is treated with ultrasound at a temperature ranging from about 36 °C to about 38 °C. In another embodiment, the ECM in a liquid is treated with ultrasound at a temperature ranging from about 37 °C or greater, such as from about 37 °C to about 55 °C, such as from about 37 °C to about 50 °C, such as from about 37 °C to about 45 °C, such as from about 37 °C to about 40 °C. The ECM in a liquid is treated with ultrasound 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 another embodiment, the ECM in a liquid is treated with ultrasound at greater than about 38 °C, such as from about 38 °C to about 50 °C, such as from about 38 °C to about 45 °C, such as from about 38 °C to about 40 °C.

[0088] The acoustic ECM hydrogel is manufactured with ultrasound. The acoustic ECM hydrogel typically undergoes a phase transition from sol to gel at around 37 °C, and thus transitions to a liquid phase at greater than 37 °C, and to a gel phase at less than 37 °C. At 37 °C, the acoustic ECM hydrogel is sufficiently viscous to resemble a gel, however when the temperature is raised above 37 °C, the gel transitions to a sol. The acoustic ECM hydrogel forms a gel (sol to gel transition) when the temperature is lowered below 37 °C. Thus, in some embodiments, after sonication, the acoustic ECM hydrogel is cooled to a temperature below 37 °C, such as from about 4 °C to about 36 °C. The acoustic ECM hydrogel can be cooled to room temperature, which is typically about 25 °C. In some embodiments, the acoustic ECM hydrogel is cooled to from about 15 °C to about 25 °C. The acoustic ECM hydrogel can be cooled to from about 23 °C to about 27 °C. The acoustic ECM hydrogel can be cooled to about 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 to induce the gel phase.

[0089] In some embodiments, an acoustic mammalian ECM hydrogel is disclosed, wherein the hydrogel is thermoreversibly reversible, wherein the hydrogel is in a solid (gel) phase at temperatures below about 37°C and in a liquid (sol) phase at temperatures above 37°C. The acoustic hydrogel can be made using any of the methods disclosed herein. In some embodiments, the acoustic ECM hydrogel has a storage modulus (G') that is about one order of magnitude greater than the loss modulus (G"). In further embodiments, the acoustic ECM hydrogel has a viscosity that decreases with increasing stress at temperatures from about 15 to about 37°C, such as at temperatures of 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 acoustic ECM hydrogel has a viscosity that decreases with increasing stress at room temperature, and / or at temperatures from about 23°C to about 27°C and / or from about 15°C to about 25°C. In one embodiment, the acoustic ECM hydrogel has a gel to sol transition at about 37°C, such that the hydrogel is sufficiently viscous at body temperature to be useful as a submucosal spacer.

[0090] The acoustic ECM hydrogels can be made from any of the mammalian ECMs disclosed above. In specific, non-limiting examples, the ECM is a human ECM. In other non-limiting examples, the ECM is a bladder ECM, a small intestine submucosa ECM, an esophageal EMC, or a dermal ECM. In one embodiment, the ECM is a bladder ECM. In another embodiment, the ECM is a dermal ECM. In yet another embodiment, the ECM is an esophageal ECM. The source of the ECM can be, for example, a pig, a cow, or a sheep.

[0091] In some embodiments, the acoustic ECM hydrogel comprises ECM at a concentration of about 25 milligrams per milliliter to about 600 milligrams per milliliter. In further embodiments, the acoustic ECM hydrogel comprises ECM at a concentration of about 20 milligrams per milliliter to about 600 milligrams per milliliter, about 25 milligrams per milliliter to about 300 milligrams per milliliter, about 25 milligrams per milliliter to about 200 milligrams per milliliter, and about 25 milligrams per milliliter to about 150 milligrams per milliliter. In further embodiments, the acoustic ECM hydrogel comprises ECM at a concentration of about 50 milligrams per milliliter to 600 milligrams per milliliter in a liquid, for example, in a buffer. The acoustic ECM hydrogel can also have an ECM concentration of about 50 milligrams per milliliter to about 300 milligrams per milliliter, about 50 milligrams per milliliter to about 200 milligrams per milliliter, about 50 milligrams per milliliter to about 150 milligrams per milliliter, about 50-100 milligrams per milliliter, or about 100-150 milligrams per milliliter. In some non-limiting examples, the acoustic ECM hydrogel comprises ECM at a concentration of about 20, 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 milligrams per milliliter. In some non-limiting examples, the acoustic ECM hydrogel comprises ECM at a concentration of about 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, 115-120, 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 milligrams per milliliter. Exemplary non-limiting concentrations of ECM also include about 25 milligrams per milliliter, 100 milligrams per milliliter, and 150 milligrams per milliliter. In one non-limiting example, the acoustic ECM hydrogel comprises ECM at a concentration of about 25 milligrams per milliliter to about 150 milligrams per milliliter. In one embodiment, the ECM concentration is about 100 milligrams per milliliter.

[0092] In some embodiments, the acoustic ECM hydrogel has a viscosity of about 1400 Pa*s at 15°C and about 400 Pa*s at 25°C when the ECM concentration is about 150 mg / mL. In other embodiments, the acoustic ECM hydrogel has a storage modulus of about 2700 Pa*s at 15°C, about 800 Pa*s at 25°C, and 600 Pa*s at 37°C when the ECM concentration is about 150 mg / mL.

[0093] Liquid-phase acoustic ECM hydrogels can be placed in a three-dimensional cast before cooling, or spread on... To form a film on the sheet, see, for example Figure 1C The high concentration of ECM (50 to 600 mg / mL) in the acoustic ECM hydrogel allows for the formation of very thin sheets, such as sheets as thin as 4 micrometers. The acoustic ECM hydrogel can be configured to any size greater than 4 micrometers and to be any 2D or 3D shape. In some embodiments, sheets with a thickness of about 4 to about 10 micrometers are formed, such as sheets with thicknesses of about 4, 5, 6, 7, 8, 9, or 10 micrometers. The acoustic ECM hydrogel can be shaped into any three-dimensional shape, including but not limited to cylinders, spheres, ellipsoids, disks, sheets, cubes, cuboids, cones, triangular or rectangular prisms, as well as hollow spheres, hollow ellipsoids, and hollow cylinders with open ends. Figures 2A-2C An exemplary shape is shown in the figure. Acoustic ECM hydrogels can also be used as injectables, for example, by placing them in a syringe and extruding them from the syringe as a gel phase or a sol phase.

[0094] In some embodiments, the acoustic ECM hydrogel is absorbed into, adsorbed onto, or otherwise dispersed onto or into a biocompatible substrate. Non-limiting examples of biocompatible substrates include: meshes, non-wovens, decellularized tissues, polymeric compositions, polymeric structures, cell growth scaffolds, implants, orthopedic implants and intraocular lenses, sutures, intravascular implants, stents, and grafts. In some embodiments, the substrate is synthetic. In other embodiments, the substrate is natural. The acoustic ECM hydrogel can be applied or incorporated into non-woven materials, such as bandages, sutures, implants, such as ceramic, metal or polymeric implants, such as prostheses, artificial or otherwise engineered vessels, valves, intraocular lenses or tissue implants, by any suitable method. As used herein, the terms "coated" and related cognates such as "coated" and "coating" refer to a process that includes partial or total coverage of an inorganic structure with a composition described herein. For example and without limitation, coating an inorganic structure with an acoustic ECM hydrogel in a liquid phase can include processes such as casting, embedding, layering, impregnating, spraying.

[0095] In another embodiment, a composition comprising an acoustic ECM hydrogel is coated onto a biocompatible structural material (such as a metal, an inorganic calcified compound such as calcium hydroxide, calcium phosphate or calcium carbonate, or a ceramic composition) in a liquid phase. Non-limiting examples of suitable metals are cobalt-chromium alloys, stainless steel alloys, titanium alloys, tantalum alloys, titanium-tantalum alloys, which can include non-metallic and metallic components such as molybdenum, tantalum, niobium, zirconium, iron, manganese, chromium, cobalt, nickel aluminum, and lanthanum, including but not limited to various grades of CP Ti (commercially pure titanium) or Ti 6A14V (90% by weight Ti, 6% by weight Al, and 4% by weight V), stainless steel 316, Nitinol (nickel-titanium alloy), titanium alloys coated with hydroxyapatite. Metals are useful due to high strength, flexibility, and biocompatibility. Metals can also be shaped into complex shapes, and many can withstand corrosion in a biological environment, reduce wear, and do not cause tissue damage. Other compositions, including ceramics, calcified compounds such as but not limited to aragonite. Combinations of metals, ceramics, and / or other materials can also be used.

[0096] Any useful agent can be mixed into, co-delivered with, co-administered with, or otherwise combined with any of the compositions described herein. For example and without limitation, useful agents include interferons, interleukins, chemokines, monokines, hormones, coagulants, chemotherapeutic agents, and antibiotics.

[0097] Methods of use

[0098] Macrophages have been shown to be important regulators in normal healing after injury and normal tissue development. The disclosed acoustic ECM hydrogels can recapitulate the effects of the whole ECM on macrophage phenotype, leading to an increase in M2-like, regulatory, or pro-remodeling macrophages. Accordingly, any of the compositions disclosed herein can be used to modify macrophage phenotype, for example, to induce regulatory M2 macrophages.

[0099] In some embodiments, a method of inducing M2 macrophages in a subject is disclosed by administering a therapeutically effective amount of a composition comprising an acoustic ECM hydrogel as disclosed herein, thereby inducing M2 macrophages in the subject. In additional embodiments, a method of reducing Ml (pro-inflammatory) macrophages in a subject is disclosed. The method comprises administering a therapeutically effective amount of an acoustic ECM hydrogel, thereby inhibiting Ml macrophages in the subject. The subject can be any contemplated subject, including human and veterinary subjects.

[0100] The disclosed acoustic ECM hydrogels increase hemostasis at a lesion in a subject. Accordingly, methods for accelerating clotting of a wound and / or reducing the bleeding time of a wound are also disclosed. In some embodiments, hemostasis is induced within about 10 to about 100 seconds, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 seconds, after administration of the acoustic ECM hydrogel to the subject.

[0101] In some embodiments, a therapeutically effective amount of an acoustic ECM hydrogel can be administered locally to a site in a subject to induce hemostasis. The subject can have a wound. The wound can be an external wound, or an internal wound that is not viable from outside the patient's body. The disclosed acoustic ECM hydrogels are useful as hemostatic agents for any type of wound. The method can comprise selecting any of the contemplated subjects, such as those having any wound.

[0102] As Figure 10As shown, the acoustic ECM hydrogel reduces clotting time. Clotting can be measured by any method known to one of skill in the art. In the Lee and White test tube method, venous blood is placed in three test tubes, held in a 37°C water bath. The time for firm clot to form in each of the first and then second test tubes is recorded sequentially by tilting them at one minute intervals, and then the third test tube is examined for clotting of the blood to obtain the clotting time, thereby determining the clotting time. In the capillary method, a glass capillary is filled with blood from a finger prick. At regular intervals, short sections of the capillary are broken off until clotting of the blood occurs between the broken sections of the capillary. Another method of determining the occurrence of clotting is the thromboelastogram method. In this method, a fork moving in a sample of blood or plasma is used to sense the viscosity, which increases upon clotting. Another method of recording the occurrence of clotting is to monitor the translucency of a plasma sample after it has been separated from the blood. As clotting occurs, the sample becomes opaque.

[0103] In some embodiments, methods for treating a subject having an inflammation or a wound are disclosed. The method includes topically applying a therapeutically effective amount of an acoustic ECM hydrogel to the inflammation or wound. In some non-limiting examples, the subject has an inflammatory condition, such as, but not limited to, ulcerative colitis or rheumatoid arthritis. The method can include applying the ECM hydrogel to a tissue surface. In other non-limiting examples, the subject is an organ transplant recipient, a subject having graft versus host disease, a subject having a myocardial infarction, or a subject having a wound, such as, but not limited to, a subject having a surgical wound or a non-surgical traumatic wound. Accordingly, methods of accelerating wound healing and / or increasing hemostasis in an individual in need thereof are disclosed, comprising administering a therapeutically effective amount of a composition comprising an acoustic ECM hydrogel as disclosed herein. The administration can be topical, for example, at the site of a wound or graft.

[0104] The hydrogel can be applied to any wound site to increase hemostasis and / or increase wound healing. The wound can be a wound in the skin, or a wound on any surface, including, but not limited to, the eye. Methods for wounds caused by ischemia and ischemic injury, such as chronic venous leg ulcers caused by impairment and / or dysfunction of venous circulation system return, are also provided. Accordingly, the methods of the present invention can utilize topical skin or ocular administration. Typically, in these administrations, the composition is formulated for topical administration. The hydrogel can be applied to a tissue surface of any organ.

[0105] Disclosed herein are topical compositions for healing wounds, such as skin wounds. These wounds can be superficial or can be deep and involve damage to the dermis and epidermis of the skin. The wounds can be surgical wounds. Thus, methods for promoting wound healing in a subject and / or for promoting coagulation (increasing hemostasis) in a subject are provided.

[0106] The acoustic ECM hydrogel can be applied directly to the target site, for example, in a topical formulation, such as a patch, plug, or as part of a dressing or bandage. The bandages and wound dressings can comprise the acoustic ECM hydrogel. These can be prepared by applying the acoustic ECM hydrogel in a gel or liquid phase to the bandage or wound dressing along with any other desired additives. These patches, plugs, bandages or dressings can be used to reduce coagulation time and / or increase wound healing. The acoustic hydrogel can be administered by injection to the target site to promote wound healing, for example, in the form of a solid in a gel phase, or the temperature can be raised above 37°C prior to administration such that the hydrogel is administered in a liquid phase.

[0107] For use in wound treatment and / or for increasing hemostasis, the acoustic ECM hydrogel will typically have a concentration in the ranges described above. The acoustic ECM hydrogel can be applied once. Alternatively, the acoustic ECM hydrogel can be applied periodically to the affected area, typically about 1 to 10 times per day, for example, over a period of about 3 to 14 days, depending on the nature of the wound. In some cases, it can be desirable to apply the composition indefinitely.

[0108] The acoustic ECM hydrogel affects the rate of hemostasis and wound healing. In some embodiments, the composition increases hemostasis and / or wound healing by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, or at least 200% compared to a control, such as a standard value, the rate of wound healing or hemostasis achieved without treatment or treatment with an ECM hydrogel manufactured by enzymatic methods.

[0109] The acoustic ECM hydrogel can also be used to treat surgical wounds and other intentional interventions, where the composition can be applied immediately after the procedure is completed. Methods for stimulating wound healing and increasing hemostasis at a wound site are provided, including surgical wounds, excisional wounds, deep wounds involving damage to the dermis and epidermis, ocular tissue wounds, dental tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow sore ulcers, arterial ulcers, venous stasis ulcers, and burns caused by heat exposure or chemicals.

[0110] The subject can be any contemplated mammalian subject, including human or veterinary subjects. The subject can be a pediatric or adult subject, e.g., a young, middle-aged, or elderly adult subject. In humans, adult subjects are greater than 18 years of age, young adults are about 18 to about 35 years of age, middle-aged adults are generally considered to be about 35 to about 55 years of age, and slightly older (or elderly) human subjects are greater than about 55 years of age, e.g., greater than 60 years of age, greater than 65 years of age, greater than 70 years of age, greater than 75 years of age, or greater than 80 years of age.

[0111] The subject can heal wounds at a normal rate or can have a healing impairment. Many painful and conditions can result in a healing impairment. These include diabetes (e.g., type II diabetes), treatment with steroids and other pharmacological agents, and ischemic blockage or injury (as in peripheral vascular disease or traumatic vascular occlusion). Conditions that induce abnormal wound healing include, but are not limited to, uremia, malnutrition, vitamin deficiency, obesity, infection, immunosuppression, and complications associated with systemic treatment with steroids, radiation therapy, and antineoplastic drugs and antimetabolites. Steroids that have been shown to impede wound healing include cortisone, hydrocortisone, dexamethasone, and methylprednisolone. Non-steroidal compounds, such as octreotide acetate, have also been shown to impede wound healing (Waddell et al., Am. Surg. 63:446 449, 1997).

[0112] The subject can have a coagulation disorder, or can be undergoing treatment with an anticoagulant such as, but not limited to, warfarin or The subject can have a deficiency in coagulation factors II, V, VII, X, or XII. The subject can have hemophilia A, hemophilia B, von Willebrand disease, a deficiency or structural abnormality of fibrinogen or prothrombin. Thus, in some embodiments, these subjects are selected for treatment.

[0113] Also provided herein are methods of increasing adhesion of a skin graft to a wound bed and stimulating re-epithelialization from a wound bed. Types of grafts include, but are not limited to, autologous skin graft, artificial skin, allografts, autodermic graft, autoepidermic grafts, avascular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. The methods include administering to a subject having a graft a therapeutically effective amount of a composition disclosed herein, thereby increasing adhesion and acceptance of the graft and controlling or eliminating bacterial growth. In some embodiments, the cell or tissue treated with the composition is transplanted into a subject. In one specific, non-limiting example, the composition is administered to a graft, e.g., a skin graft, prior to transplantation.

[0114] Methods of treating blisters and burns due to abrasion or chemical damage are also provided. These methods include treatment of the skin or internal organs. These methods include treatment of ovarian damage, for example, due to treatment with a chemotherapeutic agent or treatment with cyclophosphamide; radiation or chemotherapy-induced cystitis; or high-dose chemotherapy-induced intestinal damage. The methods include administering to a subject a therapeutically effective amount of a composition disclosed herein to promote healing of the blister or burn and to reduce or eliminate bacterial growth.

[0115] Methods for promoting healing of anastomotic wounds and other wounds resulting from surgical procedures in an individual are provided. These methods include administering an effective amount of a composition disclosed herein after and / or during an anastomosis or other surgery. An anastomosis is the joining of two tubular structures, for example, when a section of intestine is removed and the remaining portions are joined together to reestablish the intestinal tract. Unlike skin healing, the healing process of an anastomotic wound is often obscured from view. In addition, wound healing in the gastrointestinal tract, at least, proceeds rapidly in the absence of complications; however, complications often require correction by additional surgery (Thornton and Barbul, Surg. Clin. North Am. 77:549 573 (1997)). The methods can include selecting a subject in need of anastomotic wound healing. The subject can be one who has a wound healing disorder due to one of the above-mentioned conditions, or can be one who has normal wound healing, for example, a subject who does not have any of the above-mentioned conditions.

[0116] The disclosed acoustic ECM hydrogel is in a solid phase at room temperature and transitions to a liquid phase at about 37°C. Thus, in some embodiments, upon administration to a subject, as the hydrogel warms due to body temperature, the acoustic ECM hydrogel will transition from a solid phase to a liquid phase over time. In some embodiments, the method can include washing the wound to remove the acoustic ECM hydrogel, which can be rinsed away in the liquid phase.

[0117] In some embodiments, the acoustic ECM hydrogel is sterilized. Sterilization is important to ensure that the acoustic ECM hydrogel of the present application is sufficiently free of contamination by pathogens and suitable for medical applications, such as implantation into a human or animal body. Methods such as gamma irradiation, ethylene oxide, supercritical CO2, hydrogen peroxide gas plasma, or ozone can be suitable for sterilization of the acoustic ECM hydrogel of the present application, although other sterilization methods known in the art can also be suitable. As shown herein, gamma sterilization is an acceptable method of sterilization because the acoustic ECM hydrogel of the present application maintains its stiffness upon gamma sterilization. In one embodiment, the acoustic ECM hydrogel is gamma sterilized prior to the ECM solution forming a gel, e.g., the ECM in liquid can be sterilized prior to sonication or after sonication prior to forming a gel. In another embodiment, the acoustic ECM hydrogel is gamma sterilized after forming a gel. In some embodiments, the acoustic ECM hydrogel remains in gel form after sterilization by gamma irradiation, or can form a gel.

[0118] In contrast, for enzymatically manufactured hydrogels, gamma irradiation destabilizes the composition. Thus, after gamma irradiation, no gel is formed, or the gel is destabilized.

[0119] Acoustic ECM hydrogel as a submucosal spacer

[0120] Endoscopy is a procedure that allows the internal part of hollow organs or cavities of the body to be examined without resorting to invasive surgery, through an instrument called an endoscope. Endoscopy can be used for surgical procedures, such as cauterizing bleeding blood vessels, removing polyps, adenomas and small tumors, performing biopsies or removing foreign bodies. Endoscopy procedures can be performed in the gastrointestinal tract, respiratory tract, ear, urinary tract, female reproductive system, and through small incisions in normally closed body cavities, such as the abdominal cavity or pelvis (laparoscopy), inside joints (arthroscopy) and thoracic organs (thoracoscopy and mediastinoscopy). Endoscopy can be performed in the upper or lower gastrointestinal tract. An endoscope is an illuminated, usually optical fiber, flexible or rigid, tubular instrument used for diagnostic or therapeutic purposes to visualize the interior of a hollow organ or part, such as the bladder, esophagus, stomach or intestine, which usually has one or more working channels to enable the passage of instruments, such as forceps, electrosurgical knives, endoscopic injection needles or scissors, or to facilitate the removal of biopsy samples. It includes a suitable light and imaging device at its distal part, and it can be inserted through a naturally occurring opening of the body, such as the mouth, anus, ear, nose, or through a small surgical incision. Given the wide variety of body organs or cavities that can be examined by endoscopy procedures, there are several types of specialized endoscopes, such as laryngoscopes, thoracoscopes, angioscopes, colonoscopes, enteroscopes, sigmoidoscopes, rectoscopes, proctoscopes, anoscopes, arthoscopes, nasoscopes, laparoscopes, hysteroscopes, brainscopes, nephoscopes, esophagoscopes, bronchoscopes, gastroscope, amnioscopes, cystoscopes.

[0121] Endoscopy procedures are widely used in the gastrointestinal tract, including the upper and lower gastrointestinal tract. For example, endoscopy procedures can be used to examine the mucosa covering the gastrointestinal lumen and to detect small and large pathological lesions, such as inflammatory tissue, polyps, pseudopolyps, serrated lesions, adenomas, ulcers, dysplasias, pre-neoplastic and neoplastic formations, and tumors. Endoscopy procedures can be used for biopsy and removal of pathological lesions (polyps, adenomas, dysplasias, pre-neoplastic and neoplastic formations, tumors). Surgical procedures include two types of endoscopic resection procedures commonly used in gastrointestinal endoscopy to remove pathological lesions: endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). Both techniques allow the minimally invasive treatment of gastrointestinal polyps, adenomas, dysplasias and early cancers, which involves minimal risk of lymph node metastasis.

[0122] Disclosed herein are methods for dissection of mucosa and submucosa from the underlying muscularis layer of an organ region of a subject. The organ can be in the gastrointestinal tract, e.g., esophagus, duodenum, stomach, small intestine, large intestine (colon), or rectum. The organ can be the bladder, an organ of the oral-respiratory system (lungs, throat (pharynx), tongue, nasal passages, sinuses), skin, or uterus and vagina. Examples of specific tissues are respiratory epithelium, nasal epithelium, dermal or epidermal tissue, and uterine epithelium. One exemplary organ is the esophagus. Another exemplary organ is the colon. The methods are for any organ having mucosa and submucosa, where superficial lesions, e.g., malignant or premalignant lesions, can form.

[0123] The methods include submucosal injection of a pharmaceutical composition comprising an acoustic ECM hydrogel into an organ of a subject to form a cushion between the submucosa and underlying muscularis layer of a region of the organ. In one embodiment, the organ is not the esophagus. In another embodiment, the organ is the esophagus. The methods can be endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD).

[0124] EMR is an endoscopic technique that was developed for the removal of sessile or flat neoplasms that are confined to the superficial layers (mucosa and submucosa) of the gastrointestinal (GI) tract. EMR is commonly used to remove lesions smaller than 2 cm or to remove larger lesions piecemeal. EMR also plays an important role in assessing the resected specimen for accurate pathologic staging. In contrast to polypectomy, EMR involves lifting the lesion off the muscularis layer by injecting a fluid agent, typically a normal saline (NS) solution, into the submucosa. EMR can also be used to obtain a specimen for precise histopathologic staging to determine the risk of lymph node metastasis. EMR facilitates complete removal of the affected mucosa by resection through the intermediate or deeper portions of the submucosa of the intestinal wall. Various EMR techniques have been described, and four methods involving snare resection are commonly used: (1) injection and cutting method; (2) injection, lifting, and cutting method; (3) cap-assisted EMR (EMRC); and (4) EMR with ligation (EMRL). In the injection and cutting technique, the diseased mucosa is lifted from the muscularis layer by creating a submucosal fluid cushion, captured using an electrosurgical snare, and then resected. However, injection into a thin submucosa is a delicate procedure, the injected solution tends to dissipate rapidly, flat and depressed lesions are difficult to capture by the snare compared to protruding lesions, and large or awkwardly positioned lesions can be difficult to remove (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). Injection-assisted EMR is often used for large flat colonic polyps.

[0125] Endoscopic submucosal dissection (ESD) was specifically developed for the removal of larger lesions. Using a surgical electrotome, the lesion is directly dissected along the submucosa, resulting in en-bloc resection even of large lesions. ESD has been predicted to replace traditional surgery in the treatment of certain stages of cancer, but requires a greater degree of endoscopic skill and experience than EMR due to its higher rate of perforation and bleeding complications than traditional EMR. ESD can use a number of electrosurgical knives, such as an insulation-tipped diathermic knife, a needle knife, a hook knife, a flex knife, a triangle tipped knife, a flush knife, a splash needle, and a small-caliber tip transparent hood. These knives can be used with a high frequency electrosurgical current (HFEC) generator. ESD is characterized by three steps: (1) injection of fluid to create a submucosal cushion, thereby lifting the lesion from the muscularis layer; (2) circumferential cutting of the surrounding mucosa; and (3) dissection of the submucosal connective tissue under the lesion (see Kakushima et al., Wold J. Gastroenterol. 14(9): 2962-2967, 2008, incorporated herein by reference). Various submucosal injection solutions have been previously developed and shown to be satisfactory for use during EMR, but the introduction of the longer ESD procedure requires a more durable solution to help identify the cutting line during submucosal dissection (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). The presently disclosed methods meet this need.

[0126] Submucosal injection is used in EMR because injection of fluid into the submucosal cushion facilitates the separation of the tissue to be removed prior to capture of the target lesion, such as with a snare, thereby reducing the risk of thermal injury and perforation and bleeding, while also facilitating resection. Submucosal injection plays an important role in the EMR procedure because the solution must remain in place for sufficient time and needs to form a hemi-sphere to facilitate snaring. In addition, providing a high enough submucosal lift results in safe submucosal cutting during ESD procedures (Uraoka et al., Drug Design, Development and Therapy 2008:2131-138). Furthermore, because the procedure results in inflammation, any cushion that remains at the site of the procedure should have anti-inflammatory properties. The acoustic ECM hydrogel will reduce stenosis and promote epithelial reformation. The presently disclosed method also meets this need.

[0127] In some embodiments, the disclosed method utilizes an acoustic ECM hydrogel that has anti-inflammatory properties and is inexpensive, non-toxic, easy to inject, and provides a high and persistent submucosal cushion. The acoustic ECM hydrogel is administered in its gel state at the injection site to form the cushion. The cushion can be peeled off during the procedure so that some of the hydrogel remains on the underlying muscularis propria, thereby aiding healing. The disclosed acoustic ECM hydrogel promotes closure of the wound created by removal of the resected mucosa / submucosa. In some embodiments, the procedure is ESD. In other embodiments, the procedure is EMR.

[0128] Normal saline solution (NS) and diluent solutions (e.g., ELEVIEW TM , see U.S. Patent 9,226,996, incorporated herein by reference) have been used as submucosal cushions for endoscopic resection, but the inherent properties of these solutions make it difficult to create an appropriate submucosal fluid cushion, maintain the desired height, and keep the cushion in the desired location due to the rapid dispersion of the solution. In addition, in ESD, once the mucosa / submucosa is removed, these agents will not remain on the underlying muscularis propria. Furthermore, these agents do not aid the healing process, for example, by reducing inflammation. The use of an acoustic ECM hydrogel meets these needs.

[0129] The acoustic ECM hydrogel disclosed herein can be used in any ESD or ESR. As disclosed in U.S. Patent 9,364,580, incorporated herein by reference, an endoscopic injection needle is a device that can be long (up to about 230 cm) and includes a relatively long catheter, within which an inner injection tube having a distal injection needle is slidably disposed. A proximal actuation handle is connected to the catheter and the injection tube so as to move one relative to the other when desired. Fluid access to the injection tube is typically provided through a leer connector on the handle. The endoscopic injection needle device is typically delivered to the injection site through the working channel of an endoscope. To protect the lumen of the endoscope working channel from damage, the handle of the infusion needle device is manipulated to retract the distal injection needle into the lumen of the catheter prior to insertion of the device into the endoscope. This prevents exposure of the sharp tip of the injection needle as the device moves through the lumen of the endoscope. When the distal end of the endoscopic injection needle device is at the injection site, its handle is again manipulated to move the injection needle distally outside of the lumen of the catheter. The length of the exposed portion of the injection needle is about 4-6 mm when advanced to the most distal position.

[0130] After the injection site is punctured, the acoustic ECM hydrogel, typically contained in a 5 to 10 milliliter syringe fitted with a luer-lock fitting connected to the handle of the injection needle, can be delivered to the injection site, such as between the submucosa and the underlying muscularis propria, through the injection tube and needle.

[0131] The injection needle and other accessories typically used during an endoscopy procedure, such as a snare for polypectomy, a clipping device, a biopsy forceps, etc., pass through one or more specific channels of the endoscope, typically referred to as working channels or operating channels. The internal diameter of the working channels can vary significantly depending on the type of endoscope used in GI endoscopy, such as a gastroscope, enteroscope, colonoscope, duodenoscope, sigmoidoscope, etc. However, the most common endoscopes used for GI endoscopy have working channels with internal diameters in the range of about 2 mm to about 5 mm. Typically, the manufacturers of endoscopic accessories produce accessories with an external diameter that allows them to fit all working channels. In some embodiments, the endoscopic injection needle, the external diameter of the catheter ranges from 1.9 mm to 2.3 mm, such as about 1.9, 2.0, 2.1, 2.2, or 2.3 cm. Thus, considering that the inner injection tube is contained within the outer catheter, its internal diameter is typically 1 mm or less. The disclosed acoustic ECM hydrogel, in gel or liquid form, can be easily passed through these catheters.

[0132] The acoustic ECM hydrogel can be used in an endoscopic resection procedure by drawing a volume of the hydrogel from its main container through a syringe, injecting a suitable volume of the hydrogel directly under the superficial mucosal layer by an endoscopic injection needle inserted into the working channel of an endoscope to deposit the hydrogel into the submucosa, which becomes a cushion upon being in place: the elevation of the mucosal surface allows the endoscopist to easily resect a mucosal lesion found during the performance of an endoscopy procedure, even if the lesion is flat and thus does not protrude into the lumen such as the intestinal, esophageal or gastric cavity. At body temperature, the acoustic ECM hydrogel is a viscous but flowable gel that transitions into a liquid phase and can be easily injected under the superficial mucosal layer to form a cushion for the procedure. Because the gel-sol transition takes time, the cushion remains in place for enough time to perform the resection.

[0133] The presence of at least one dye in the cushion can help the endoscopist visualize the structures underneath the mucosa (e.g. the submucosa and the external muscularis wall), thus reducing the risk of damage to said structures by the endoscopist performing the resection procedure. The use of a dye can allow visualization of the cushion cavity and the mucosal base. Removal of the lesion from the mucosal surface creates a mucosal wound. The persistence of the cushion created by the injected volume of the pharmaceutical composition allows the endoscopic resection procedure to be performed without the need for re-injection. The acoustic ECM hydrogel is injected submucosally into the area of interest of the subject’s organ, for example in the area of a lesion or tumor, to form a cushion between the submucosa and the underlying muscularis propria at the area of the organ. The cushion can be stripped such that a portion of the acoustic ECM hydrogel remains on the underlying muscularis propria and aids in the healing process.

[0134] The disclosed methods are for the esophagus. In one non-limiting example, the method comprises a method of stripping an esophageal carcinoma or adenocarcinoma from the esophagus. In another non-limiting example, the method comprises stripping the mucosa and submucosa from the esophagus of a subject having a Barrett’s esophagus. In these embodiments, the acoustic ECM hydrogel can be a bladder, small intestinal submucosa (SIS), esophagus, trachea, liver, or skin acoustic ECM hydrogel.

[0135] The disclosed methods are also used for other organs. The organ can be any contemplated organ, such as an organ of the gastrointestinal tract. The organ can be in the upper gastrointestinal tract, such as the pharynx, tongue, or mouth. The organ can be the bladder, vagina, or uterus. In some embodiments, the organ is the colon, duodenum, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine. In one non-limiting example, the organ is the stomach, small intestine, or large intestine, and the method comprises a method of debulking a carcinoma or adenocarcinoma from the stomach. In another non-limiting example, the organ is the colon, and wherein the method comprises debulking a polyp or carcinoma from the colon. In these embodiments, the acoustic ECM hydrogel can be a bladder, small intestinal submucosa, esophageal, tracheal, liver, or skin acoustic ECM hydrogel.

[0136] The acoustic ECM hydrogel as disclosed herein is maintained at a temperature at which it gels, and thus is administered in the form of a submucosal pad, at or below that temperature.

[0137] The acoustic ECM hydrogel can be maintained at, for example, about 4°C or about room temperature prior to administration. In one embodiment, the acoustic ECM hydrogel can be administered at a temperature of, for example, 4°C to less than 37°C, or 4°C to 25°C. In one embodiment, the acoustic ECM hydrogel is administered at a temperature less than 37°C. An effective amount of the acoustic ECM hydrogel as a gel is then used. The acoustic ECM hydrogel remains as a gel in the tissue of the subject at a temperature of about 37°C. In one embodiment, the gel-to-sol transition of the acoustic ECM hydrogel is at about 37°C, such that the hydrogel is useful as a submucosal pad because it is sufficiently viscous at body temperature.

[0138] In some embodiments, the concentration of ECM in the hydrogel is from 25 mg / ml to about 200 mg / ml, such as about 25 mg / ml to about 100 mg / ml of ECM hydrogel. In other embodiments, the concentration of ECM in the hydrogel is from about 50 to about 150 mg / ml, such as about 75 to about 125 mg / ml, such as about 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 mg / ml. In one particular non-limiting example, the concentration of ECM in the hydrogel is about 100 mg / ml.

[0139] The acoustic ECM hydrogel can be provided in lyophilized form at room temperature, cold temperature (e.g., about 4°C), or frozen (e.g., about -20°C), and reconstituted just prior to administration to the contemplated anatomical region of the subject.

[0140] The disclosed methods are for any subject, including human and veterinary subjects. The subject can be of any age. The subject can be an adult or a juvenile. In one embodiment, a composition comprising an acoustic ECM hydrogel is injected into a target tissue in an organ to form a cushion, which is then optionally subjected to an endoscopic surgical procedure, such as a resection procedure. The ECM can be from the same species as the subject being treated, or can be from a different species. In some embodiments, the subject is a human, and the acoustic ECM hydrogel is derived from human or porcine ECM. In other embodiments, the ECM hydrogel is derived from non-human primate, dog, cat, horse, or bovine. The acoustic ECM can also be from a commercial source. In some embodiments, the acoustic ECM hydrogel can be derived from any mammalian tissue, such as but not limited to porcine or human tissue, and in some non-limiting examples, can be derived from the bladder, small intestine, or esophagus. Any of the above disclosed acoustic ECM hydrogels derived from any source tissue can be used as a submucosal cushion and / or in any of the disclosed methods. The acoustic ECM hydrogel can be an esophageal acoustic ECM hydrogel or a bladder acoustic ECM hydrogel.

[0141] The disclosed methods are invasive in that they require injection of the mucosa and submucosa away from the native muscle layer of the organ region of the subject's gut. In some embodiments, the acoustic ECM hydrogel is not applied to the surface of the organ (e.g., organ of the gastrointestinal tract, such as the esophagus). The disclosed methods can be used for the esophagus, but can also be used for other tissues.

[0142] Any of the methods disclosed herein can include injecting a pharmaceutical composition comprising an acoustic ECM hydrogel from the submucosa into an organ of a subject to form a cushion between the submucosa and the underlying muscularis of the organ region. Suitable acoustic ECM hydrogels are disclosed above. The acoustic ECM hydrogel gels and peels the mucosa and submucosa away from the underlying muscularis and suppresses inflammation in the organ region in the subject. The acoustic ECM hydrogel, as a gel, can be administered through an endoscope or through a catheter. In some embodiments, the organ is the esophagus, colon, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine. The acoustic ECM hydrogel, as a gel or sol, can also be administered through an endoscope or through a catheter. In further embodiments, the acoustic ECM hydrogel can be a bladder, small intestinal submucosa, esophageal, tracheal, liver, or skin acoustic ECM hydrogel. In some embodiments, the ECM can be from human tissue. In other embodiments, the ECM can be from porcine tissue.

[0143] In some embodiments, the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection, and the method comprises a method of dissection of esophageal carcinoma or adenocarcinoma from the esophagus. In further embodiments, the method comprises dissection of the mucosa and submucosa from the esophagus of a patient having a developmental abnormality. In further embodiments, the method comprises dissection of the mucosa and submucosa from the esophagus of a subject having Barrett’s esophagus.

[0144] In some embodiments, the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection. In further embodiments, the organ is the stomach, small intestine, or large intestine, and the method comprises a method of dissection of a polyp, carcinoma, or adenocarcinoma from the colon. In further embodiments, the method comprises dissection of the mucosa and submucosa from the organ of a patient having a developmental abnormality. In a specific, non-limiting example, the method comprises dissection of a polyp or carcinoma from the colon.

[0145] The method can further comprise performing the endoscopic resection procedure on the bolster. In some embodiments, the method comprises separating the bolster such that the hydrogel remains on the lower layer of the intrinsic muscle layer of the esophagus, and removing the mucosa and submucosa from the region of the esophagus. In some non-limiting examples, the portion of the hydrogel bolster that remains on the lower layer of the intrinsic muscle layer downregulates proinflammatory macrophage activation in the esophagus.

[0146] The disclosure is illustrated by the following non-limiting examples.

[0147] Example

[0148] Current methods of ECM to fabricate hydrogels involve digestion of ECM material with acid proteases in acidic solutions (Feyetes, Biomaterials 29(11) (2008) 1630-7; Voytik-Harbin, Tissue Engineering 4(2) (1998) 157-174), digestion with alpha-amylase to fabricate ECM foams (Kommuller et al., JoVE (Journal of Visualized Experiments) (122) (2017) e55436); or use of chaotropic extraction buffers and long dialysis procedures (Uriel, Tissue Eng Part C Methods 15(3) (2009) 309-21; Uriel, Biomaterials 29(27) (2008) 3712-9). ECM hydrogels prepared according to this method inevitably undergo protein degradation and denaturation, which can diminish the full complement of ECM molecules and the bioactivity of tissue-specific ECM components. Furthermore, enzyme-based methods of fabricating ECM hydrogels require long incubation times of 24-72 hours to achieve sufficient solubilization of ECM components, and require the addition of exogenous enzymes for digestion (Saldin et al., Acta Biomater 49 (2017) 1-15; Spang et al., Acta Biomaterialia 68 (2018) 1-14). ECM hydrogels prepared using enzyme digestion are also hindered by limited concentration-dependent rheological properties (Saldin et al., supra, 2017). To realize the full clinical potential of ECM hydrogels, fundamentally different methods were developed whereby ECM hydrogels can be rapidly formed without the use of acidic or basic solutions, protease digestion, or chemical extraction and dialysis.

[0149] Methods of manufacturing acoustic ECM hydrogels using ultrasonic cavitation are disclosed, as well as characterization of the viscoelastic properties, cell compatibility, and bioactivity of these acoustic ECM hydrogels. In some embodiments, using pulverized ECM as a starting material, the method includes resuspending the ECM in a neutral buffered saline solution, and then solubilizing using a 20 kHz ultrasonic frequency. Rapid gelation of the ECM solution can be induced by reducing the temperature of the ECM solution to a temperature below 25 °C. The gelation time and ECM gel properties can be easily adjusted by modulating the ECM concentration, and the amplitude and time of sonication. Once polymerized, the ECM gel is stable at temperatures ranging from 4 °C to 37 °C. Furthermore, the ECM hydrogels prepared using this method are biocompatible and are able to promote an M2-like, pre-remodeling macrophage phenotype, which is beneficial for downstream constructive tissue remodeling (Hussey et al., Nature Reviews Materials, 3: 159-173 (2018)). These methods offer the advantage of large-scale manufacturing of acoustic ECM hydrogels compared to traditional enzymatic methods of manufacturing hydrogels with different properties. The acoustic ECM hydrogels manufactured by the presently disclosed methods can be used for clinical applications based on tissue engineering and regenerative medicine.

[0150] The present invention relates to the following scheme:

[0151] Scheme 1. A method of manufacturing an extracellular matrix (ECM) hydrogel, comprising solubilizing a mammalian ECM in a liquid using an ultrasonic frequency to manufacture an acoustic ECM hydrogel in a liquid phase.

[0152] Scheme 2. The method of scheme 1, wherein the ultrasonic frequency is applied at a temperature between 30 °C and 43 °C.

[0153] Scheme 3. The method of scheme 1 or 2, wherein the ultrasonic frequency is applied at a temperature between 35 °C and 40 °C.

[0154] Scheme 4. The method of any one of schemes 1-3, wherein the ultrasonic frequency is applied at a temperature between 36 °C and 38 °C.

[0155] Scheme 5. The method of any one of schemes 1-4, wherein the ultrasonic frequency is applied at a temperature of about 37 °C.

[0156] Scheme 6. The method of any one of schemes 1-5, wherein the mammalian ECM is present at a concentration of about 25 milligrams per milliliter to about 600 milligrams per milliliter.

[0157] Scheme 7. The method of any one of schemes 1-6, wherein the ultrasonic frequency is 20 kHz or greater.

[0158] Scheme 8. The method of any one of schemes 1-7, wherein the ultrasonic frequency is 20 kHz to about 100 kHz.

[0159] Scheme 9. The method of any one of schemes 1-8, wherein the ultrasound frequency is applied for at least 30 seconds.

[0160] Scheme 10. A method of manufacturing an extracellular matrix (ECM) hydrogel, comprising sonicating a mammalian ECM in a liquid at a concentration of about 25 milligrams per milliliter to about 600 milligrams per milliliter at a temperature greater than about 37 °C with ultrasound at a frequency of about 20 kHz to about 100 kHz for at least about 30 seconds to manufacture an acoustic ECM hydrogel in a liquid phase.

[0161] Scheme 11. The method of any one of schemes 1-10, wherein the mammalian ECM is treated with ultrasound for at least about 60 seconds.

[0162] Scheme 12. The method of any one of schemes 1-11, wherein the mammalian ECM is a lyophilized mammalian ECM.

[0163] Scheme 13. The method of scheme 12, wherein the lyophilized mammalian ECM is pulverized into a powder.

[0164] Scheme 14. The method of any one of schemes 1-13, wherein the ECM is provided in the form of fragments ranging from about 10 μιη to about 2000 μιη.

[0165] Scheme 15. The method of any one of schemes 1-13, wherein the ECM is provided in the form of fragments ranging from about 10 μιη to about 1000 μιη.

[0166] Scheme 16. The method of any one of schemes 1-15, further comprising cooling the acoustic ECM hydrogel in a liquid phase to a temperature of about 37 °C or less, thereby manufacturing an acoustic ECM hydrogel in a gel phase.

[0167] Scheme 17. The method of scheme 16, wherein the cooling is to a temperature of 4 °C to 25 °C.

[0168] Scheme 18. The method of scheme 16, wherein the cooling is to a temperature of 4 °C to less than 37 °C.

[0169] Scheme 19. The method of any one of schemes 1-18, wherein the frequency of the ultrasound is about 20 kHz.

[0170] Scheme 20. The method of any one of schemes 1-19, wherein the ultrasound has an amplitude of about 20 μιη to about 320 μιη.

[0171] Scheme 21. The method of scheme 20, wherein the ultrasound has an amplitude of about 36 μιη to about 180 μιη.

[0172] Scheme 22. The method of any one of schemes 1-21, comprising treating the solubilized mammalian ECM in a liquid with ultrasound for about 60 seconds to about 1 hour.

[0173] Protocol 23. The method of any of protocols 1-22, comprising treating the solubilized mammalian ECM in the liquid with ultrasound for about 60 seconds.

[0174] Protocol 24. The method of any of protocols 1-23, comprising providing the mammalian ECM in the liquid at a concentration of 25 milligrams per milliliter to 150 milligrams per milliliter.

[0175] Protocol 25. The method of any of protocols 1-24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 30-45 °C.

[0176] Protocol 26. The method of any of protocols 1-24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 35 °C to 40 °C.

[0177] Protocol 27. The method of any of protocols 1-24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 36 °C to 38 °C.

[0178] Protocol 28. The method of any of protocols 1-24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 37 °C.

[0179] Protocol 29. The method of any of protocols 1-24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 40 °C.

[0180] Protocol 30. The method of any of protocols 1-29, wherein the ECM is a human ECM.

[0181] Protocol 31. The method of any of protocols 1-30, wherein the liquid is phosphate buffered saline.

[0182] Protocol 32. The method of any of protocols 1-31, wherein the ECM is a bladder ECM, a small intestine submucosa ECM, an esophagus ECM, a trachea ECM, a liver ECM, or a skin ECM.

[0183] Protocol 33. The method of any of protocols 1-32, wherein the ECM is a porcine ECM or a bovine ECM.

[0184] Protocol 34. The method of any of protocols 1-33, wherein the ECM is a porcine esophagus ECM.

[0185] Protocol 35. The method of any of protocols 1-33, wherein the ECM is a porcine bladder ECM.

[0186] Protocol 36. The method of any of protocols 1-35, further comprising placing the acoustic ECM hydrogel in the liquid phase into a three-dimensional cast prior to cooling.

[0187] Scheme 37. The method of scheme 36, wherein the cast produces a sheet that is at least 4 microns thick.

[0188] Scheme 38. The method of any one of schemes 1-37, wherein the acoustic ECM hydrogel is gamma irradiated.

[0189] Scheme 39. The method of scheme 38, wherein the acoustic ECM hydrogel is gamma irradiated at about 20 kGy.

[0190] Scheme 40. An acoustic ECM hydrogel made by the method of any one of schemes 1-39.

[0191] Scheme 41. An acoustic ECM hydrogel, wherein the hydrogel is thermoreversible, wherein the hydrogel is in a gel phase at temperatures below about 37 °C and in a liquid phase at temperatures above about 37 °C.

[0192] Scheme 42. The acoustic ECM hydrogel of any one of schemes 40 or 41, wherein the storage modulus (G') is about an order of magnitude greater than the loss modulus (G").

[0193] Scheme 43. The acoustic ECM hydrogel of any one of schemes 40-42, wherein the viscosity of the acoustic ECM hydrogel decreases with increasing stress at temperatures from about 15 °C to about 37 °C.

[0194] Scheme 44. The acoustic ECM hydrogel of any one of schemes 40-43, wherein the mammalian ECM hydrogel is a human ECM hydrogel.

[0195] Scheme 45. The acoustic ECM hydrogel of any one of schemes 40-44, wherein the ECM is a bladder ECM, a small intestine submucosa ECM, an esophagus ECM, a trachea ECM, a liver ECM, or a skin ECM.

[0196] Scheme 46. The acoustic ECM hydrogel of any one of schemes 40-45, wherein the ECM is a porcine ECM.

[0197] Scheme 47. The acoustic ECM hydrogel of any one of schemes 40-46, wherein the acoustic ECM hydrogel has a viscosity of about 1400 Pa*s at 15 °C and a viscosity of about 400 Pa*s at a temperature of 25 °C, and wherein the acoustic ECM hydrogel comprises an ECM at a concentration of about 150 milligrams per milliliter.

[0198] Scheme 48. The acoustic ECM hydrogel of any one of schemes 40-47, wherein the acoustic hydrogel has a storage modulus of about 2700 Pa*s at 15 °C, about 800 Pa*s at 25 °C, and 600 Pa*s at 37 °C, and wherein the acoustic hydrogel comprises ECM at a concentration of about 150 milligrams / milliliter.

[0199] Scheme 49. The acoustic ECM hydrogel of any one of schemes 40-48, wherein the hydrogel is gamma irradiated.

[0200] Scheme 50. The acoustic ECM hydrogel of any one of schemes 40-49, wherein the hydrogel does not contain an exogenous protease or an inactivated exogenous protease.

[0201] Scheme 51. The acoustic ECM hydrogel of any one of schemes 40-50, wherein the hydrogel does not contain an exogenous pepsin, trypsin, or hyaluronidase or an inactivated form of an exogenous pepsin, trypsin, or hyaluronidase.

[0202] Scheme 52. A method of increasing hemostasis at a lesion in a subject, comprising locally administering to the lesion a therapeutically effective amount of the acoustic ECM hydrogel of any one of schemes 40-51, whereby hemostasis is increased.

[0203] Scheme 53. The method of scheme 52, wherein the lesion is a surgical wound, a burn, or a traumatic injury.

[0204] Scheme 54. The method of scheme 52 or 53, wherein the subject is a human.

[0205] Scheme 55. The method of any one of schemes 52-54, wherein the lesion is in a blood vessel, a liver, a lung, or a skin of the subject.

[0206] Scheme 56. The method of any one of schemes 52-55, wherein hemostasis is induced within about 10 to about 100 seconds after the acoustic ECM hydrogel is administered to the subject.

[0207] Scheme 57. A method of inducing an M2 phenotype in a macrophage, comprising treating the macrophage with an effective amount of the acoustic ECM hydrogel of any one of schemes 40-51, whereby the M2 phenotype is induced.

[0208] Scheme 58. The method of scheme 57, wherein the subject is a human.

[0209] Scheme 59. A method for delaminating a mucosa and submucosa from a native muscle layer of an organ region of a subject, comprising:

[0210] submucosally injecting a pharmaceutical composition comprising the acoustic ECM hydrogel of any one of schemes 40-51 into an organ of the subject to form a spacer between the submucosa and the underlying native muscle layer of the organ region,

[0211] thereby detaching the mucosa and submucosa from the underlying muscularis layer and suppressing inflammation in the organ region in the subject.

[0212] Scheme 60. The method of scheme 59, wherein the acoustic ECM hydrogel is fabricated from bladder ECM, small intestine submucosa ECM, esophagus ECM, trachea ECM, liver ECM, or skin ECM.

[0213] Scheme 61. The method of scheme 59 or scheme 60, wherein the concentration of ECM in the acoustic ECM hydrogel is 25 milligrams per milliliter to about 600 milligrams per milliliter.

[0214] Scheme 62. The method of scheme 59 or scheme 60, wherein the concentration of ECM in the acoustic ECM hydrogel is 25 milligrams per milliliter to about 100 milligrams per milliliter.

[0215] Scheme 63. The method of any one of schemes 59-62, wherein the acoustic ECM hydrogel is administered through an endoscope or through a catheter.

[0216] Scheme 64. The method of any one of schemes 59-63, wherein the organ is the esophagus, stomach, colon, rectum, or small intestine.

[0217] Scheme 65. The method of scheme 64, wherein the colon is the ascending colon, transverse colon, descending colon, or sigmoid colon.

[0218] Scheme 66. The method of scheme 64, wherein the small intestine is the jejunum, cecum, or ileum.

[0219] Scheme 67. The method of any one of schemes 59-66, wherein the method comprises a method of detaching an adenocarcinoma or carcinoma from an organ.

[0220] Scheme 68. The method of scheme 67, wherein the organ is the stomach, small intestine, or colon.

[0221] Scheme 69. The method of any one of schemes 64-65, wherein the organ is the colon and the method

[0222] Scheme 70. The method of any one of schemes 64, 65, or 69, wherein the organ is the colon, and wherein the method comprises detaching a polyp or carcinoma from the colon.

[0223] Scheme 71. The method of any one of schemes 59-64, wherein the organ is the esophagus, and wherein the method comprises detaching the mucosa and submucosa from the muscularis layer of the esophagus.

[0224] Scheme 72. The method of scheme 71, wherein the subject has Barrett’s esophagus.

[0225] Scheme 73. The method of any one of schemes 59-72, further comprising performing an endoscopic resection procedure on the lining to remove the stripped mucosa and submucosa.

[0226] Scheme 74. The method of scheme 73, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection.

[0227] Scheme 75. The method of scheme 74, wherein the method comprises:

[0228] Separating the lining such that the acoustic ECM hydrogel remains on the underlying muscularis propria layer of the organ, and removing the mucosa and submucosa from the organ region.

[0229] Scheme 76. The method of any one of schemes 59-75, wherein the subject is a human.

[0230] Scheme 77. The method of any one of schemes 59-76, wherein the organ is in the gastrointestinal tract.

[0231] Example 1

[0232] Materials and Methods

[0233] Preparation of dermal ECM: Dermal ECM was prepared as previously described (Reing JE, et al. Biomaterials. 2010;31(33):8626-33). Briefly, full-thickness skin was harvested from market weight (approximately 110 kg) pigs (Tissue Source Inc.) and subcutaneous fat and epidermis were removed by mechanical delamination. The tissue was then treated with 0.25% trypsin (Thermo Fisher Scientific) for 6 hours, 70% ethanol for 10 hours, 3% H2O2 for 15 minutes, 1% Triton X-100 (Sigma-Aldrich) / 0.26% EDTA / 0.69% tris for 6 hours, solution change for an additional 16 hours, and 0.1% peracetic acid / 4% ethanol (Rochester Midland) for 2 hours. Water washes were performed between each chemical change, with alternating water and phosphate buffered saline (PBS) washes after the final step. All chemical exposures were performed with agitation at 300 rpm on an orbital shaker. The dermal ECM was then lyophilized and ground into particulates using a Wiley mill with a #60 mesh screen.

[0234] Urinary bladder matrix (UBM) preparation: UBM was prepared as previously described (Mase VJ, et al. Orthopedics. 2010;33(7):511). Porcine bladders from market weight animals were obtained from Tissue Source, LLC. Briefly, the serosal, outer muscular, submucosal, and muscularis mucosae layers were mechanically removed. The luminal urothelial cells of the mucosa were detached from the basement membrane by washing with deionized water. The remaining tissue consisted of the basement membrane and the underlying lamina propria layer of the mucosa and was decellularized by agitation in 0.1% peracetic acid with 4% ethanol at 300 rpm for 2 hours. The tissue was then rinsed extensively with PBS and sterile water. The UBM was then lyophilized and ground into particles using a Wiley mill with a #60 mesh screen.

[0235] Small intestinal submucosa (SIS) preparation: SIS was prepared as previously described (Badylak SF, et al. J Surg Res. 1989;47(1):74-80). Briefly, jejunal segments were harvested from 6-month-old market weight (about 110 to about 120 kg) pigs and split longitudinally. The superficial layers of the mucosa were mechanically removed. Likewise, the serosal and outer muscular layers were mechanically removed, leaving the submucosal and basal portions of the mucosa. Decellularization and sterilization of the tissue was accomplished by agitation in 0.1% peracetic acid with 4% ethanol at 300 rpm for 2 hours. The tissue was then rinsed extensively with PBS and sterile water. The SIS was then lyophilized and ground into particles using a Wiley mill with a #60 mesh screen.

[0236] Preparation of esophageal ECM: Esophageal ECM (eECM) was prepared as previously described (Keane TJ, et al. Tissue Eng Part A. 2015;21(17-18):2293-300). Briefly, esophageal ECM was prepared by mechanically separating the mucosa and submucosa from the outer muscularis and subjecting the mucosal layer to the following conditions: 1% trypsin / 0.05% EDTA (Invitrogen, Carlsbad, CA) for 1 hour at 37°C on a rocker plate, deionized water for 15 minutes, 1 M sucrose (Fisher Scientific, Pittsburgh, PA) for 30 minutes, deionized water for 30 minutes, 3.0% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 48 hours, deionized water for 15 minutes, phosphate buffered saline (PBS; Fisher Scientific) for 15 minutes, 10% deoxycholate (Sigma-Aldrich) for 4 hours, deionized water for 30 minutes, 0.1% peracetic acid (Rochester Midland Corp., Rochester, NY) / 4.0% ethanol for 4 hours, 100 U / mL DNAse (Invitrogen) for 2 hours on a rocker plate, followed by washes with PBS, deionized water, PBS, and deionized water for 15 minutes. All washes were agitated on a shaker plate at 300 rpm. The ECM of the esophagus was then lyophilized and ground into particles using a Wiley mill with a #60 mesh screen.

[0237] Ultrasonication of ECM: 100 mg of ECM powder was resuspended in phosphate buffered saline (PBS) in a 15 mL conical tube and sonicated using a FISHERBRAND TM 120-type Sonic Dismembrator equipped with a 1 / 8" probe. The concentration of ECM varied from 20-200 mg (w / v). The resuspended ECM was sonicated with 30 seconds on and 45 seconds off cyclic pulses at 100% amplitude setting. This cycle was repeated six times to produce a soluble ECM solution. This cyclic pulse setting ensured that the gel solution was kept at a temperature in the range of 34-40°C.

[0238] Gelation of sonicated ECM solution: The ECM solution was poured into a 3D mold and the temperature was reduced to 25°C or lower to induce gelation. The ECM gel was stored at 4°C or lyophilized to produce a lyophilized ECM construct that retains its 3D geometry. Alternatively, the ECM solution was uniformly spread on a Teflon sheet and incubated at 4°C to induce gelation. The ECM gel was then incubated at room temperature for 24 hours to evaporate water, producing an ultrathin ECM sheet.

[0239] Preparation of ECM putty: ECM powder was resuspended in 25 mg / ml (w / v) PBS and sonicated as described above. At concentrations < 25 mg / ml and temperatures between 4-30 °C, the solubilized ECM formed putty.

[0240] Rheology of ECM hydrogels: All rheological data were collected using a rheometer (AR2000, TA instruments, New Castle, DE) equipped with a 40 mm parallel plate geometry as previously described (Medberry CJ, et al. Biomaterials. 2013;34(4): 1033-40) and analyzed using the American Society for Testing and Materials (ASTM) standard F2900-11 (Guidelines for the characterization of hydrogels for regenerative medicine). Temperature was controlled to within 0.1 °C using a Peltier plate. Gel precursors were loaded onto the parallel plate rheometer at room temperature (25 °C) or 4 °C. The edges of the sample-plate interface were sealed using mineral oil to minimize sample evaporation.

[0241] Scanning electron microscopy: Scanning electron micrographs were taken to examine the surface topography of ECM hydrogels. Samples were fixed in cold 2.5% (v / v) glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) / PBS for at least 24 hours and then washed three times in PBS. Fixed samples were then dehydrated using a graded series of alcohols (30, 50, 70, 90, 100%) for 15 minutes each, followed by 15 minutes in hexamethylenediamine (Fisher) and then air dried. Dried samples were sputter coated with a 3.5 nm layer of gold / palladium alloy using a sputter coater 108 Auto (Cressington Scientific Instruments, Watford, UK) and imaged at 100x and 500x magnification using a JEOL JSM6330f scanning electron microscope (JEOL, Peabody, MA).

[0242] Cell compatibility analysis: 3T3 fibroblasts were seeded onto 96-well plates coated with ECM hydrogels prepared from UBM, SIS, or dermis. Uncoated wells were used as controls. Cells were cultured in Dulbecco’s Modified Minimal Essential Medium provided with 10% fetal bovine serum and 1% penicillin-streptomycin. Twenty-four hours after seeding the cells, cell viability was determined using the MTT Cell Proliferation Assay Kit (Thermo Fisher) according to the manufacturer’s protocol. Absorbance of the converted dye was measured at a wavelength of 540 nm. MTT cell proliferation assay kit (Thermo Fisher) according to the manufacturer’s protocol. Absorbance of the converted dye was measured at a wavelength of 540 nm.

[0243] Hemostasis analysis. Lee White clot analysis was used. Fresh whole blood was collected into a test tube and the test tube was repeatedly tilted until clotting was observed.

[0244] Liver laceration model: 6-8 week old Sprague-Dawley rats were anesthetized with isoflurane (1-3%). The animals were maintained in the surgical plane of anesthesia with 1.5-2.5% isoflurane in oxygen and the animals were placed in a ventral recumbency position. Using sterile instruments, a small incision was made and a 2 cm midline laparotomy was performed extending underlying to expose the liver. A 2 mm incision was made on the ventral surface of the liver by placing a #11 scalpel blade in a Kelly clamp such that 2 mm of the blade was exposed, and a 2 mm deep and 5 mm long incision was made. The wound was allowed to bleed for 3 seconds and then wiped clean with a sterile gauze. The test article was then placed at the site of the defect and the clotting time was recorded.

[0245] Macrophage activation: Murine bone marrow was harvested from 6-8 week old B6 mice. Cells harvested from the bone marrow were washed and plated at 2 x 106cells / ml and allowed to differentiate into macrophages for 7 days in the presence of macrophage colony stimulating factor (MCSF) with complete media changed every 48 hours. The macrophages were then activated for 24 hours with one of the following: 1) 20 ng / ml interferon-gamma (IFNy) and 100 ng / ml lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) to promote the MIFNy+LPS phenotype (Ml-like); 2) 20 ng / ml interleukin (IL)-4 (Invitrogen) to promote the MIL-4 phenotype (M2-like); or 3) 2 mg / ml UBM Acoustic Gel. After the 37°C incubation period, the cells were washed with sterile PBS and the cells were fixed with 2% paraformaldehyde (PFA) for immunolabeling. To prevent non-specific binding, the cells were incubated in blocking solution consisting of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin for 1 hour at room temperature. The blocking buffer was then removed and the cells were incubated in primary antibodies. The cells were incubated at 4°C for 16 hours, the primary antibodies were removed, and the cells were washed with PBS. A solution of fluorophore-conjugated secondary antibodies was added to the wells for 1 hour at room temperature. The antibodies were 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 thereafter kept constant in all groups.

[0246] Example 2

[0247] Results

[0248] A method for preparing hydrogels from extracellular matrix (ECM) was developed. Using decellularized tissue as starting material, sonication techniques can be applied to a wide range of tissue-specific ECMs, including dermis, urinary bladder matrix (UBM), and small intestinal submucosa (SIS). The method includes resuspending the pulverized ECM in a neutral buffered saline solution, followed by ECM lysis using, for example, a 20 kHz ultrasonic frequency, using an amplitude of 20-100% (Fig. 1). Rapid gelation of the ECM solution is induced by reducing the temperature of the ECM solution to below 37°C after 60 seconds of sonication (Figs. 4, 5, 6). Results of rheological evaluation show that using this method, ECM hydrogels can be prepared using ECM concentrations ranging from 25 mg / ml to 150 mg / ml Figure 7 , Figure 8 ). Once polymerized, the ECM gel is stable at room temperature and can be conformed to customizable 3D geometries (Fig. 2). ECM hydrogels prepared by sonication show cell compatibility when used as a substrate for culturing cells in vitro (Fig. 9, Figure 13 ). Scanning electron micrographs of the gel show a dense fibrillar network Figure 3 . In addition, ECM hydrogels can be used as a hemostatic agent, which can be applied to an anatomical site of a patient to aid in hemostasis Figure 10 , Figure 11 . Results are provided in the accompanying figures.

[0249] Example 3

[0250] Materials and methods for Examples 4-7

[0251] Preparation of ECM bioscaffolds: Porcine skin ECM (dECM) was prepared as previously described (Reing et al., Biomaterials 31(33) (2010) 8626-33). Briefly, full-thickness skin was harvested from market weight (approximately 110 kg) pigs and subcutaneous fat and epidermis were removed by mechanical delamination. The tissue was then treated with 0.25% trypsin (Thermo Fisher Scientific) for 6 hours, 70% ethanol for 10 hours, 3% H2O2 for 15 minutes, 1% Triton X-100 (Sigma-Aldrich) / 0.26% EDTA / 0.69% tris for 6 hours, with solution change for an additional 16 hours, and 0.1% peracetic acid / 4% ethanol (Rochester Midland) for 2 hours. Water washes were performed between each chemical change, with alternating water and phosphate buffered saline (PBS) washes after the final step. All chemical exposures were performed with agitation at 300 rpm on an orbital shaker. The skin ECM was then lyophilized and ground into a particulate using a Wiley mill with a #40 mesh screen.

[0252] Porcine urinary bladder matrix (UBM) was prepared as previously described (Mase et al., Orthopedics 33(7):511 (2010)). Briefly, the serosal, outer muscular, submucosal, and muscularis mucosae layers were mechanically removed. The luminal urothelial cells of the mucosa were detached from the basement membrane by washing with deionized water. The remaining tissue consisted of the basement membrane and the underlying lamina propria layer of the mucosa and was decellularized by agitation in 0.1% peracetic acid with 4% ethanol at 300 rpm for 2 hours. The tissue was then rinsed extensively with PBS and sterile water. The UBM was then lyophilized and ground into particles using a Wiley mill with a #40 mesh screen.

[0253] Porcine small intestinal submucosa (SIS) was prepared as previously described (Badylak et al., J Surg Res 47(1) (1989) 74-80). Briefly, the jejunum was harvested from a 6-month-old market weight (about 110 to about 120 kg) pig and split longitudinally. The superficial layer of the mucosa was mechanically removed. Likewise, the serosal and outer muscular layers were mechanically removed, leaving the submucosal and basal portions of the mucosa. Decellularization and sterilization of the tissue was accomplished by agitation in 0.1% peracetic acid with 4% ethanol at 300 rpm for 2 hours. The tissue was then rinsed extensively with PBS and sterile water. The SIS was then lyophilized and ground into particles using a Wiley mill with a #40 mesh screen.

[0254] Pig esophageal ECM was prepared as previously described (Keane et al., Tissue Eng Part A 21(17-18) (2015) 2293-300). Briefly, esophageal ECM (eECM) was prepared by mechanically separating the mucosa and submucosa from the outer muscularis and subjecting the mucosal layer to the following conditions: 1% trypsin / 0.05% EDTA (Invitrogen, Carlsbad, CA) for 1 hour at 37 °C on a rocker plate, deionized water for 15 minutes, 1 M sucrose (Fisher Scientific, Pittsburgh, PA) for 30 minutes, deionized water for 30 minutes, 3.0% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 48 hours, deionized water for 15 minutes, phosphate buffered saline (PBS; Fisher Scientific) for 15 minutes, 10% deoxycholate (Sigma-Aldrich) for 4 hours, deionized water for 30 minutes, 0.1% peracetic acid (Rochester Midland Corp., Rochester, NY) / 4.0% ethanol for 4 hours, 100 U / mL DNAse (Invitrogen) for 2 hours on a rocker plate, followed by washes with PBS, deionized water, PBS, and deionized water for 15 minutes. All washes were agitated on a shaker plate at 300 rpm. The eECM was then lyophilized and ground into particles using a Wiley mill with a #40 mesh screen.

[0255] Pig tracheal ECM (tECM) was prepared with slight modifications as previously described (Lange et al., Journal of tissue engineering and regenerative medicine 11(3) (2017) 800-811). Briefly, trachea was incubated in a detergent solution containing 0.25% Triton X-100 + 0.25% sodium deoxycholate for 30 minutes of negative pressure vacuum cycling (15 cycles, -0.95 kPa maximum vacuum), then soaked in fresh detergent solution overnight. This process was repeated daily, replacing the detergent solution with sterile DI water on the second and third days, 2000 KU / mL DNase aqueous solution on the fourth day, and sterile DI water on the fifth day. This cycle was repeated once for a total of 10 days of vacuum cycling, then sterilized in 15% peracetic acid + 4% ethanol overnight and washed and stored in sterile PBS. The tracheal ECM was then lyophilized and ground into particles using a Wiley mill with a #40 mesh screen.

[0256] Porcine liver ECM (LECM) was prepared as previously described (Loneker et al., Journal of Biomedical Materials Research Part A 104(4) (2016) 957-965). Livers were harvested from market weight pigs (110-130 kg). The tissue was cut into 0.5 cm 3 pieces and washed in deionized water for three 15 minute washes on an orbital shaker with mechanical agitation. Sections were then gently massaged to aid in cell lysis and soaked in 0.02% trypsin / 0.05% EGTA for 2 hours at 37°C. The tissue was rinsed with Type 1 water and massaged again, followed by mechanical agitation of the liver sections in 3% Triton X-100 for 18-24 hours. Rinsing was repeated until all visible cell material was removed. After processing, the liver ECM was immersed in a 0.1% peracetic acid solution and then rinsed repeatedly in Type 1 water or PBS at pH 7.4. The liver ECM was then lyophilized and ground into particles using a Wiley Mill with a #40 mesh screen.

[0257] Dissolution of ECM by ultrasonic cavitation: The ground ECM was resuspended in 10 mL of IX Phosphate Buffered Saline (PBS) in a 50 mL conical tube and sonicated with a FISHERBRAND TM Model 120 Sonic Dismembrator equipped with a 1 / 8" probe. The ECM concentration varied from 25 to 100 mg / mL (w / v) and the sonication time varied from 30 to 500 seconds with an amplitude ranging from 20% - 100%. Figure 14 shows a schematic of the experimental setup.

[0258] Quantification of collagen and sGAG: Sonicated ground dECM (100 mg / mL) and the samples were centrifuged at 10,000 x g for 30 minutes to compact the insoluble ECM components. The clear supernatant containing the solubilized ECM components was transferred to a new test tube. The collagen concentration in the supernatant solution was determined using the Sircol Collagen Assay Kit (Biocolor Ltd. UK) following the manufacturer's recommended protocol. The sulfated glycosaminoglycan (sGAG) concentration was determined using the Blyscan Sulfated Glycosaminoglycan Assay Kit (Biocolor Ltd. UK) following the manufacturer's recommended protocol.

[0259] Gelling analysis: The gelling time was measured using the test tube inversion method (Quin et al., Frontiers in chemistry 6 (2018); El-Fiqi et al., Acta biomaterialia 9(12) (2013) 9508-9521). Immediately after sonicating sample material (25, 50, or 100 mg / ml), 0.5 ml of sample was transferred to a test tube and incubated at a constant temperature of 4°C or 25°C. The flowability of the sample was observed by inverting the test tube every minute. The time at which the sample stopped flowing was taken as the gelling time and the value was recorded.

[0260] Scanning electron microscopy: Scanning electron micrographs were taken to examine the surface topography of dECM hydrogels at 50 and 100 mg / ml. Samples were fixed in cold 2.5% (v / v) glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) / PBS for at least 24 hours, then washed three times in PBS. The fixed samples were then dehydrated using a graded series of alcohols (30, 50, 70, 90, 100%) for 15 minutes each, followed by 15 minutes in hexamethylenediamine, and then air dried. The dried samples were sputter coated with a 3.5 nm layer of gold / palladium alloy using a sputter coater 108 Auto (Cressington Scientific Instruments, Watford, UK), and imaged with a JEOL JSM6330f scanning electron microscope (JEOL, Peabody, MA).

[0261] Viscoelastic measurements: All rheological data were collected using a rheometer (AR2000ex, TA instruments, New Castle, DE) equipped with a 40 mm parallel plate geometry as previously described (Medberry et al., Biomaterials 34(4) (2013) 1033-40) and analyzed using the American Society for Testing and Materials (ASTM) standard F2900-11 (Guidelines for the characterization of hydrogels for regenerative medicine). Samples (dECM and eECM, 100 mg / ml) of each tissue type were placed at the starting temperature (4, 25, or 37 °C) for a respective temperature profile for 1 hour prior to testing. Samples were loaded onto the AR-2000ex rheometer fitted with a 40 mm parallel plate geometry set to the starting temperature. The edges of the sample-plate interface were sealed using mineral oil to minimize evaporation during testing. An oscillatory time sweep was performed for 1 hour to measure the hydrogel gelation kinetics of sonication by applying a small 0.5% oscillatory strain at a frequency of 1 rad / s and rapidly changing the temperature (37 or 4 °C) according to the temperature profile of the test. Data were exported using Trios software (TA Instruments) and analyzed using Prism v8 software (GraphPad, San Diego, CA). The "average storage modulus" was the storage modulus G' averaged over the last 10 minutes of the 60-minute test, representing the G' plateau. The time to reach 50% gelation was determined as the time to reach 50% of the average storage modulus.

[0262] In vitro metabolic analysis: 3T3 fibroblasts were seeded on 96-well plates coated with 100 mg / ml ECM hydrogels prepared from UBM, SIS, or dECM. Uncoated wells were used as controls. Cells were cultured in Dulbecco's Modified Minimal Essential Medium provided with 10% fetal bovine serum and 1% penicillin-streptomycin. Twenty-four hours after seeding the cells, cell viability was evaluated using the MTT Cell Proliferation Assay Kit (Thermo Fisher) according to the manufacturer's protocol. The absorbance of the converted dye was measured at a wavelength of 540 nm. MTT cell proliferation assay kit (Thermo Fisher) according to the manufacturer's protocol. The absorbance of the converted dye was measured at a wavelength of 540 nm.

[0263] In vitro cell compatibility: Primary equine mesenchymal stem cells were isolated as previously described (Adams et al., Equine veterinary journal 45(3) (2013) 372-375). Cells were seeded on 6-well plates coated with 100 mg / ml ECM hydrogel prepared from UBM or dECM. Uncoated wells were used as a control. Twenty-four hours after seeding, in vitro cell compatibility was determined using the Live / Dead Viability / Cytotoxicity Kit (Invitrogen) according to the manufacturer's instructions. Five 200X range images were taken in 3 technical replicates. The percentage of live and dead cells was quantified using CellProfiler. Images were taken with a Zeiss Axiovert microscope, capturing five random fields at 200X magnification. Quantification of live and dead cell percentages was done using a custom CellProfiler pipeline.

[0264] In vitro macrophage response: Murine bone marrow cells were harvested from 6-8 week old B6 mice. Cells harvested from bone marrow were washed and plated at 2 x 10 6 cells / ml and allowed to differentiate into macrophages in the presence of macrophage colony stimulating factor (MCSF) for 7 days, changing complete media every 48 hours. Macrophages were then activated for 24 hours with one of the following: 1) 20 ng / ml interferon-gamma (IFNy) and 100 ng / ml lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) to promote M IFNγ+LPS phenotype (Ml -like); 2) 20 ng / ml interleukin (IL)-4 (Invitrogen) to promote M IL-4 phenotype (M2-like); 3) 2 mg / ml dECM hydrogel; or 4) 2 mg / ml eECM hydrogel. Following a 37°C 24 hour incubation period, cells were washed with sterile PBS and cells were fixed with 2% paraformaldehyde (PFA) for immunolabeling. To prevent non-specific binding, cells were incubated in blocking solution consisting of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin for 1 hour at room temperature. Blocking buffer was then removed and cells were incubated with primary antibodies. Cells were incubated at 4°C for 16 hours, primary antibodies were removed, and cells were washed with PBS. A solution of fluorophore-conjugated secondary antibodies was added to the wells for 1 hour at room temperature. Antibodies were then removed, cells were washed with PBS, and cell nuclei were counterstained using DAPI. Cytokine-activated macrophages (positive control) were used to establish a standardized exposure time (positive control) to image the remaining treatment groups.

[0265] Statistical methods: All analyses were performed using Prism software (GraphPad Software Inc) with significance defined as p < 0.05. Collagen and sGAG results were analyzed using analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test. Gelling time results were analyzed using analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test. For single comparisons, student's unpaired t-test was performed. Rheological data were analyzed by two-way analysis of variance (ANOVA) for the independent variables temperature and ECM type and the dependent variable storage modulus, for the main effect of temperature, using post hoc Tukey's multiple comparison test. Student's unpaired t-test was performed to compare gelling times of dECM and eECM.

[0266] Example 4

[0267] Dissolution of collagen and sGAG

[0268] To evaluate the effect of sonication amplitude on the dissolution of collagen and sulfated glycosaminoglycans (sGAG), pulverized dECM was sonicated at 20%, 40%, 60%, 80%, and 100% amplitude for 300 seconds. Results showed that the dissolved collagen significantly increased with increasing sonication amplitude ( Figure 15A ). In contrast, sonication amplitude had no significant effect on the dissolution of sGAG ( Figure 15B ). To evaluate the effect of sonication time on the dissolution of collagen sGAG, pulverized dECM was sonicated at 100% amplitude for a time period of 30 to 500 seconds. Results showed that the dissolved collagen significantly increased with increasing sonication time ( Figure 15C ). In contrast, sonication time had no significant effect on the dissolution of sGAG ( Figure 15D ).

[0269] Example 5

[0270] Gelling kinetics and qualitative evaluation

[0271] The effect of sonication time and amplitude on the gelling kinetics of dECM hydrogels prepared at 25, 50, or 100 milligrams / milliliter concentration was calculated. Results showed that incubating the pre-gel solution at 4°C significantly reduced the time required to form a gel compared to 25°C for all concentrations tested ( Figure 16A ). Furthermore, 100 milligrams / milliliter concentration showed a significant reduction in gelling time compared to 25 milligrams / milliliter concentration when incubated at 4°C (Figure 16A ). Increasing the sonication amplitude from 20 or 40% to 80 or 100% amplitude resulted in a significant decrease in gelation time for all tested concentrations Figure 16B ). Significant differences in gelation time were observed between 25 and 100 mg / ml concentrations sonicated at 40 or 100% amplitude Figure 16B ). The gelation kinetics of UBM, SIS, eECM, tECM, and LECM prepared at 100 mg / ml concentration were determined Figure 16C ). The results showed that incubation of the pre-gel solution at 4°C significantly decreased the time required to form a gel for all tested ECM tissue types compared to incubation at 25°C. In addition, eECM hydrogels showed a significant decrease in gelation time at 4°C compared to all other ECM tissue types. When incubated at 25°C, eECM showed a significant decrease in gelation time compared to tECM and LECM. Scanning electron micrographs of 50 and 100 mg / ml dECM hydrogels showed a dense fibrillar network with organized collagen fibrils Figure 3

[0272] Example 6

[0273] Rheology measurements

[0274] Two tissue types of ECM hydrogels (dECM and eECM, 100 mg / ml) were tested under 3 temperature profiles: 1) 4→37°C, 2) 25→37°C, and 3) 37→4°C Figure 17A ). The ECM hydrogels showed an S-shaped increase in gelation (storage modulus, G') when the temperature was decreased (37→4°C). The hydrogels showed a storage modulus G' > loss modulus G" at the plateau of the S-shaped gelation curve. The stiffness of the hydrogels remained over time (G' » G") when the temperature was increased (4→37°C or 25→37°C) Figure 17A ). The storage modulus G' of dECM hydrogels increased when the final temperature was rapidly decreased 37→4°C (3447.3 ± 3340.1 Pa) compared to when the final temperature was increased 4→37°C (234.4 ± 215.7 Pa) (p = 0.04) or 25→37°C (245.8 ± 94.5 Pa) (p = 0.04) Figure 17B ). The stiffness of eECM also tended to increase when the final temperature was rapidly decreased 37→4°C (2237.4 ± 227.1 Pa) compared to 4→37°C (733.0 ± 363.7 Pa) or 25→37°C (624.1 ± 133.5 Pa), but was not significant (p = 0.4) Figure 17B ). The gelation time (time to 50% gelation) of the S-shaped gelation curve was determined for dECM and eECM 37→4°C​Figure 18C ) by a student's unpaired t-test (p = 0.006). No gelation time was determined for the temperature profile ramping up to 37°C, as gelation was maintained.

[0275] Example 7

[0276] In vitro cellular responses

[0277] MTT cell proliferation assays showed that ECM hydrogels prepared from dECM, UBM, or SIS were non-cytotoxic to NIH 3T3 fibroblasts ( Figure 18A ). Similarly, results from live / dead assays showed that primary equine mesenchymal stem cells remained nearly 100% viable when seeded on ECM hydrogels prepared from dECM or UBM ( Figure 18B , C). There were no differences in proliferation and viability between these treatments and when compared to cells cultured on tissue culture plastic for 24 hours (control) Figure 18B It has been previously shown that ECM hydrogels prepared using a pepsin digestion method promote an M2-like macrophage phenotype (Huleihel et al., "Macrophage phenotype in response to ECM bioscaffolds," Seminars in immunology, Elsevier, 2017, pp. 2-13; Sicari et al., Biomaterials 35 (30) (2014) 8605-8612; Dziki et al., Journal of biomedical materials research Part A 105 (1) (2017) 138-147). To evaluate whether ECM hydrogels prepared using ultrasonic cavitation methods exhibit similar effects on macrophages, primary murine bone marrow-derived macrophages were stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) to induce an Ml -like macrophage phenotype, with interleukin-4 (IL-4) to induce an M2-like phenotype, dECM hydrogel, or eECM hydrogel. All experimental groups showed uniform F4 / 80 staining. Controls showed the expected increase in iNOS when macrophages were treated with IFNγ / LPS, and an increase in Fizzl when treated with IL-4 Figure 18B , D, E). It was found that both dECM and eECM hydrogel treatments promoted M2-like macrophage activation, similar to IL-4 treated macrophages, as shown by Fizzl expression accompanied by low iNOS expression Figure 18B , D, E).

[0278] Thus, the gelation kinetics, rheological properties, and cellular compatibility and bioactivity of ECM hydrogels prepared using ultrasonic cavitation were evaluated. Although this study focused primarily on using dECM to develop and evaluate the ultrasonic cavitation method, ECM from five other source tissues were used in selectivity analyses to show that the ultrasonic cavitation method can be applied to ECM derived from decellularized tissues of each of the categories outlined in Table 1.

[0279] Table 1: Overview of ECM source tissues, and selectivity analyses used to evaluate the ultrasonic cavitation method for manufacturing ECM hydrogels. The ECM tissue sources evaluated were dermal ECM (dECM), esophageal ECM (eECM), urinary bladder matrix (UBM), small intestinal submucosa (SIS), tracheal ECM (tECM), and liver ECM (LECM).

[0280]

[0281] In the present study, ECM scaffolds were solubilized in acidic solutions without the need for digestion with acidic proteases; or using chaotropic extraction buffers and dialysis procedures, which can have an adverse effect on the molecular composition of ECM. Sonicated ECM self-assembled into a gel when incubated at temperatures equal to or below 25°C. Without being bound by theory, gelation can be due to the presence of self-assembling molecules such as collagen. In fact, sonication of ECM scaffolds resulted in a significant increase in solubilized collagen as sonication time and amplitude increased. Unlike ECM hydrogels prepared using pepsin digestion, which remain in a liquid state at 25°C and gel at 37°C, hydrogels prepared using ultrasonic cavitation methods formed stable gels when the temperature was reduced to 25°C or below. This thermomechanical property of sonicated ECM is similar to that reported for hydrolyzed collagen, which is able to form a gel upon cooling to temperatures below 30°C (Tosh et al., Applied Physics Letters 84(21) (2004) 4242-4244). However, recent studies on collagen extracted from bovine tendon using circular dichroism analysis, atomic force microscopy, and FTIR showed that the triple helical structure of collagen was not affected by sonication and remained intact (Li et al., Sonochemistry 16(5) (2009) 605-609). Similarly, collagen extracted from the skin of Lateolabrax japonicus showed that sonication at 80% amplitude for 3 hours did not induce detectable changes in the structural integrity of collagen molecules (Kim et al., Fisheries science 79(5) (2013) 849-856). In the present study, scanning electron micrographs of sonicated ECM showed a dense fibrillar network with organized collagen fibrils. Furthermore, although there was an inverse relationship between the gelation of ECM hydrogels manufactured using ultrasonic cavitation and temperature, the stiffness of the hydrogels was maintained over time (G'»G") when the temperature was increased (4→37°C or 25→37°C). These findings suggest that the gelation process of sonicated ECM is not simply a product of collagen chemistry, but rather the result of interactions between various components in solubilized ECM, including other self-assembling molecules such as laminin and proteoglycans.

[0282] NIH 3T3 fibroblasts and primary equine mesenchymal stem cells were able to adhere to and proliferate on ECM hydrogels fabricated using ultrasonic cavitation. Furthermore, although the mechanisms of action of ECM-mediated tissue remodeling are only partially understood, it has been shown that the activation status of infiltrating macrophages at the site of remodeling from a pro-inflammatory Ml -like phenotype to a constructive and pre-remodeling M2-like macrophage phenotype is a predictor of favorable downstream remodeling outcomes (Brown et al., Acta Biomater 8(3) (2012) 978-87). The results provided herein demonstrate that ECM hydrogels fabricated using ultrasonic cavitation retain the ability to promote M2-like macrophage phenotypes.

[0283] Example 8

[0284] Gamma irradiation of acoustic hydrogels

[0285] Acoustic hydrogels (100 mg / ml) were sterilized with 20 kGy gamma irradiation at room temperature. The hydrogel "stiffness" of gamma irradiated (20 kGy) and unsterilized control acoustic hydrogels (skin ECM 100 mg / ml) was measured as a function of time. The storage modulus ("stiffness") (G') and loss modulus (G") were measured by applying a small 0.5% oscillatory strain to the sample. Three temperature profiles were tested: a rapid increase in temperature from an initial storage temperature to a final temperature: 4 to 37°C, 25 to 37°C, or 37 to 4°C. Figure 20A Typical plots of time scans are shown in Figure 20B The average storage and loss moduli averaged over the last 5 minutes of the test are shown.

[0286] After the acoustic hydrogel was formed, the gel was placed in a cesium-137 irradiator and subjected to ionizing radiation at 2065 rads / min for 16 hours at room temperature, resulting in a final radiation dose of 20 kGy.

[0287] Surprisingly and unexpectedly, gamma irradiation did not affect the ability of acoustic hydrogels to remain in gel form. In contrast, enzymatically fabricated ECM hydrogels could not remain in gel form when gamma irradiated, and pre-gels of gamma irradiated ECM hydrogels could not form a gel when gamma irradiated prior to gelation.

[0288] Example 9

[0289] Acoustic hydrogels as submucosal fluid spacer

[0290] In 50 ml conical tubes, acoustical hydrogels evaluated as submucosal spacer were prepared by resuspending 1 gram of dermal ECM (dECM) powder or esophageal ECM (eECM) powder (prepared as described in Example 3) in 10 ml of phosphate buffered saline (PBS). FISHERBRAND TM 120 Sonic Dismembrator at 100% amplitude for 3 minutes. After sonication, the samples were transferred to 5 ml syringes and incubated at 4°C to induce gel formation.

[0291] Anesthesia was induced with acepromazine (0.01 mg / kg, SC) and ketamine (5-11 mg / kg) and maintained with 1-5% Isofluorane via endotracheal tube for surgical plane anesthesia. Intravenous lactated Ringer's solution was administered at 2 ml / kg / hr to the pigs throughout the procedure and immediately postoperatively. Temperature was controlled by warm water recirculating heating pads placed under the animals. Physiological parameters such as heart, respiratory rate, body temperature and responsiveness were monitored throughout the procedure. Antibiotic prophylaxis with cefazolin 25 mg / kg was administered prior to the start of the procedure.

[0292] Pigs were placed in a supine position and the mucosa of the tubular organ was evaluated using a Pentax EG3430K endoscope. After determining the reference points in the organ, the acoustical ECM hydrogel in gel form, which was dyed blue to provide visual contrast, was injected into the submucosal space at 8 mg / ml by using an Olympus Injectorforce 4 mm 23G needle, separating the mucosa and submucosa from the underlying layers at the resection sites. Approximately 2-5 ml of blue gel was injected per site. A full circumferential length of 5 cm of mucosa (100%) was removed using band ligation EMR technique. For EMR, a Cook Duette kit with ligation bands was used. The mucosa was then resected with a snare. Results are shown in Figures 19A-19C .

[0293] In view of the many possible embodiments to which the principles of our invention can be applied, it should be recognized that the illustrated embodiments are merely examples of our invention and should not be taken as limiting the scope of our invention. Rather, the scope of our invention is defined by the claims that follow. We therefore claim all that falls within the range and spirit of these claims as our invention.

Claims

1. An acoustic extracellular matrix (ECM) hydrogel, wherein the acoustic ECM hydrogel is thermoreversibly, wherein the acoustic ECM hydrogel is in a gel phase at temperatures below about 37°C and in a liquid phase at temperatures above about 37°C, wherein the acoustic ECM hydrogel is manufactured by dissolving a mammalian ECM in a liquid using ultrasound at a frequency of 20 kHz to 100 kHz, wherein the concentration of extracellular matrix in the hydrogel is 25-600 milligrams per milliliter and wherein the term "about" means within 5% of the listed value.

2. The acoustic ECM hydrogel of claim 1, wherein the storage modulus (G') is greater than the loss modulus (G") by an order of magnitude.

3. The acoustic ECM hydrogel of claim 1 or 2, wherein the viscosity of the hydrogel decreases with increasing stress at temperatures from 15°C to 37°C.

4. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel is prepared from a bladder ECM, a small intestine submucosa ECM, an esophagus ECM, a trachea ECM, a liver ECM, or a skin ECM.

5. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel is prepared from a pig, a cow, or a sheep ECM.

6. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel has a viscosity of about 1400 Pa*s at 15°C and a viscosity of about 400 Pa*s at a temperature of 25°C, and wherein the acoustic ECM hydrogel comprises ECM at a concentration of about 150 milligrams per milliliter, wherein the term "about" means within 5% of the listed value.

7. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel has a storage modulus of about 2700 Pa*s at 15°C, about 800 Pa*s at 25°C, and 600 Pa*s at 37°C, and wherein the acoustic ECM hydrogel comprises ECM at a concentration of about 150 milligrams per milliliter, wherein the term "about" means within 5% of the listed value.

8. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel is gamma irradiated.

9. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel does not contain an exogenous protease or an inactivated exogenous protease.

10. The acoustic ECM hydrogel of claim 1 or 2, wherein the hydrogel does not contain an exogenous pepsin, trypsin, or hyaluronidase or an inactivated form of an exogenous pepsin, trypsin, or hyaluronidase.

11. The acoustic ECM hydrogel of claim 1, wherein the concentration of extracellular matrix in the hydrogel is 25-300 milligrams per milliliter.

12. The acoustic ECM hydrogel of claim 1, wherein the concentration of extracellular matrix in the hydrogel is 25-200 milligrams per milliliter.

13. The acoustic ECM hydrogel of claim 1, wherein the concentration of extracellular matrix in the hydrogel is 25-100 milligrams per milliliter.

14. The acoustic ECM hydrogel of claim 1, wherein the concentration of extracellular matrix in the hydrogel is 50-150 milligrams per milliliter.

15. The acoustic ECM hydrogel of claim 1, wherein the concentration of extracellular matrix in the hydrogel is 75-125 milligrams per milliliter.

16. The acoustic ECM hydrogel of claim 2, wherein the concentration of extracellular matrix in the hydrogel is 25-300 milligrams per milliliter.

17. The acoustic ECM hydrogel of claim 2, wherein the concentration of extracellular matrix in the hydrogel is 25-200 milligrams per milliliter.

18. The acoustic ECM hydrogel of claim 2, wherein the concentration of extracellular matrix in the hydrogel is 25-100 milligrams per milliliter.

19. The acoustic ECM hydrogel of claim 2, wherein the concentration of extracellular matrix in the hydrogel is 50-150 milligrams per milliliter.

20. The acoustic ECM hydrogel of claim 2, wherein the concentration of extracellular matrix in the hydrogel is 75-125 milligrams per milliliter.

21. The acoustic ECM hydrogel of any one of claims 11 to 20, wherein the hydrogel does not contain an exogenous protease or an inactivated exogenous protease.

22. The acoustic ECM hydrogel of any one of claims 11 to 20, wherein the hydrogel does not contain an exogenous pepsin, trypsin, or hyaluronidase or an inactivated form of an exogenous pepsin, trypsin, or hyaluronidase.

23. The acoustic ECM hydrogel of any one of claims 11 to 20, wherein the hydrogel is prepared from a skin ECM, a bladder ECM, a small intestine ECM, or an esophagus ECM.

Images