Acoustic extracellular matrix hydrogels and their uses

Abstract

This invention provides a method for forming a mammalian acoustic extracellular matrix (ECM) hydrogel without the use of acidic or alkaline solutions or protease digestion, as well as the resulting acoustic ECM hydrogel. [Solution] A method for producing an extracellular matrix (ECM) hydrogel is provided, comprising using ultrasonic frequencies to solubilize mammalian ECM in a liquid to produce a liquid-phase acoustic ECM hydrogel.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the interests of U.S. Provisional Application No. 62 / 817,787, filed March 13, 2019, and U.S. Provisional Application No. 62 / 950,565, filed December 19, 2019, which are incorporated fully herein by reference.

[0002] Areas of disclosure This application relates to the field of hydrogels, and in particular to acoustic ECM hydrogels produced from mammalian extracellular matrix (ECM) using ultrasound, and their uses. [Background technology]

[0003] background Hydrogels composed of purified ECM components such as collagen, hyaluronic acid, silk fibroin, laminin, and fibronectin are widely used in tissue engineering applications. However, these purified single-component ECM biomaterials lack the complex biochemical features of natural tissue ECM. Decellularization of whole tissues or organs provides an alternative method for harvesting ECM that preserves the biochemical features of natural tissue ECM. A major advance in the use of ECM is the ability to form hydrogels, thereby extending the clinical applicability of ECM. Known techniques for producing hydrogels from ECM are largely concentrated in the digestion of ECM material by acidic proteases in acidic solutions; the use of α-amylase digestion to produce ECM forms; or the use of chaotropic extraction buffers and cumbersome dialysis procedures. ECM hydrogels produced by such techniques inevitably undergo proteolysis and denaturation, thereby weakening the bioactivity of the ECM molecules and the overall tissue-specific ECM components. Furthermore, enzyme-based methods for generating ECM hydrogels require long incubation times of 24–72 hours to achieve sufficient solubilization of ECM components. A method is needed to form ECM hydrogels without using acidic or alkaline solutions or protease digestion. [Overview of the Initiative] [Means for solving the problem]

[0004] Summary of Disclosure This specification discloses methods for producing biocompatible mammalian acoustic extracellular matrix (ECM) hydrogels. These methods include solubilizing mammalian ECM in a liquid, such as a buffered saline solution, at a concentration of 25 mg / ml to 600 mg / ml for a sufficient amount of time at a temperature of 30 to 43°C using ultrasound with a frequency of about 20 kHz to about 100 kHz, thereby producing a liquid-phase acoustic ECM hydrogel. In some embodiments, the method includes cooling the liquid-phase acoustic ECM hydrogel to a temperature of 37°C or below to produce a gel-phase acoustic ECM hydrogel. Further embodiments disclose acoustic ECM hydrogels produced using the disclosed methods.

[0005] Thermoreversible acoustic ECM hydrogels are also disclosed. In some non-limiting examples, the acoustic ECM hydrogels are solid at temperatures below approximately 37°C and liquid at temperatures above approximately 37°C. These hydrogels are produced from mammalian ECM.

[0006] Methods for using these acoustic ECM hydrogels are also disclosed.

[0007] The above and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which will proceed with reference to the accompanying drawings. [Brief explanation of the drawing]

[0008] [Figure 1]Figures 1A-1D show the preparation of acoustic ECM hydrogels using sonication. (A) Grinded dermal ECM in a 15 ml conical tube. (B) After resuspending the ECM powder in PBS, the conical tube is placed in an ice bath and the sonicator probe is inserted into the tube. (C, D) After solubilization of the ECM with sonication pulses, the pre-gel solution is pipetted into a 3D mold or spread thinly on a Teflon sheet and incubated at a temperature of ≤37°C to induce gelation. [Figure 2] Figures 2A-2D show representative images of acoustic ECM hydrogels, lyophilized gels, ultrathin ECM sheets, and ECM putty. (A) Acoustic ECM hydrogels are cast as cylinders. (B) Lyophilized acoustic ECM hydrogels maintain their 3D structure. (C) Ultrathin ultrasonic ECM sheets prepared by casting ECM gel on a Teflon sheet. (D) ECM putty prepared by ultrasonic treatment of ECM at a concentration of less than 25 mg / ml. [Figure 3] Figure 3 shows a scanning electron microscope (SEM) image. The ECM hydrogel prepared by sonication exhibited a woven, fibrous surface. [Figure 4] Figures 4A-4B show flow sweeps. 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. Steady-state stress was applied to the gel, and the resulting deformation was measured. The data show that the viscosity of the gel decreases with increasing stress, suggesting a shear-low viscosity material. [Figure 5] Figures 5A-5B show the time sweep test. The time sweep test was performed on a 50 mg / ml acoustic ECM gel 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). The data show that storage > loss modulus at all temperatures; i.e., it maintains the quality of the hydrogel. [Figure 6A]Figures 6A–6C show representative graphs of the storage modulus, loss modulus, and complex viscosity of a 50 mg / ml acoustic ECM hydrogel. The data are plotted on a log-log scale of angular frequency measured at 25°C (A), 4°C (B), or rapidly reduced from 37°C to 4°C (C) by applying a small 0.5% vibrational tension. The data show that G' is approximately an order of magnitude larger than G'', indicating that this material meets the criteria for a hydrogel. [Figure 6B] Figures 6A–6C show representative graphs of the storage modulus, loss modulus, and complex viscosity of a 50 mg / ml acoustic ECM hydrogel. The data are plotted on a log-log scale of angular frequency measured at 25°C (A), 4°C (B), or rapidly reduced from 37°C to 4°C (C) by applying a small 0.5% vibrational tension. The data show that G' is approximately an order of magnitude larger than G'', indicating that this material meets the criteria for a hydrogel. [Figure 6C] Figures 6A–6C show representative graphs of the storage modulus, loss modulus, and complex viscosity of a 50 mg / ml acoustic ECM hydrogel. The data are plotted on a log-log scale of angular frequency measured at 25°C (A), 4°C (B), or rapidly reduced from 37°C to 4°C (C) by applying a small 0.5% vibrational tension. The data show that G' is approximately an order of magnitude larger than G'', indicating that this material meets the criteria for a hydrogel. [Figure 7] Figure 7 shows the 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 the viscosity of the gel decreases with increasing stress in the concentration range between 25–150 mg / ml and the temperature range between 15–37°C, suggesting shear-low viscosity material. Shear-low viscosity means that viscosity decreases with increasing flow (for example, the faster you "push" the material through an opening such as a needle or syringe end, the "easier" it is to pass the material through the opening, which is advantageous for clinical applications). [Figure 8]Figure 8 shows the time sweep test. To determine the maximum G' (storage modulus) and G'' (loss modulus) values ​​of the acoustic hydrogel, time sweep tests were performed at three different concentrations: 25, 100, and 150 mg / ml at 15°C, 25°C, or 37°C. The data show that storage > loss modulus at all temperatures and all concentrations; i.e., it maintains the quality of the hydrogel. [Figure 9A] Figures 9A-9B show the cytocompatibility assay. (A) 3T3 fibroblasts were seeded on petri dishes coated with control (uncoated) or acoustic ECM hydrogel prepared from UBM, SIS, or dermis, and cultured for 24 hours. The VYBRANT® MTT cell proliferation assay kit (Thermo Fisher) was used to assess cell viability. The results show that all ECMs were non-cytotoxic to 3T3 fibroblasts (n=3). (B) Viability / death assay. Hydrogel-coated plates were seeded with equine mesenchymal stem cells and compared to cells growing on tissue culture plastic. Viability was assessed using the viability / death assay kit (Invitrogen). Images were taken for five 200× fields across three technical replicates. Percent viable and dead cells were quantified using a cell profiler. Error bars represent standard deviation. [Figure 9B]Figures 9A-9B show the cytocompatibility assay. (A) 3T3 fibroblasts were seeded on petri dishes coated with control (uncoated) or acoustic ECM hydrogel prepared from UBM, SIS, or dermis, and cultured for 24 hours. The VYBRANT® MTT cell proliferation assay kit (Thermo Fisher) was used to assess cell viability. The results show that all ECMs were non-cytotoxic to 3T3 fibroblasts (n=3). (B) Viability / death assay. Hydrogel-coated plates were seeded with equine mesenchymal stem cells and compared to cells growing on tissue culture plastic. Viability was assessed using the viability / death assay kit (Invitrogen). Images were taken for five 200× fields across three technical replicates. Percent viable and dead cells were quantified using a cell profiler. Error bars represent standard deviation. [Figure 10] Figure 10 shows that the Leewhite coagulation method was used to determine the coagulation time (hemostasis) of hemostatic powders AVITENE® and XENMATRIX® ECM prepared as hydrogels by an acoustic method. XENMATRIX® is an ECM product taken from porcine dermis. The data show that AVITENE® and XENMATRIX® gels achieved rapid hemostasis compared to the untreated sample. [Figure 11] Figure 11 shows the in vivo evaluation of coagulation time using a rat liver laceration model. Rats were induced with liver lacerations and treated with hemostatic agents. SD rats were randomly assigned to five experimental groups (n=5 per group): Arista powder (BD / CR Bard), AVITENE® powder (BD / CR Bard), Micromatrix powder (ACell); indicated concentrations of esophageal ECM prepared as a hydrogel using an acoustic method; and indicated concentrations of XenMatrix (BD / CR Bard) prepared as a hydrogel using an acoustic method. The data demonstrate that mammalian ECM prepared as a hydrogel using an acoustic method can induce hemostasis in vivo. [Figure 12A]Figures 12A–12C show that acoustic hydrogels can be prepared by sonicating an ECM at a frequency of 20 kHz using amplitudes ranging from 20–100%. All samples were 50 mg / mL and were sonicated for 10 minutes before the experiment was performed at 15°C. (A) Images of the hydrogels formed at the indicated amplitudes. (B) Flow viscosity. The hydrodynamic data show that the viscosity of the gel decreases with more stress at all amplitudes tested, suggesting a shear-low viscosity material. (C) Time sweep. The hydrodynamic data show that storage > loss modulus at all concentrations at all amplitudes; i.e., it maintains the quality of the hydrogel. [Figure 12B] Figures 12A–12C show that acoustic hydrogels can be prepared by sonicating an ECM at a frequency of 20 kHz using amplitudes ranging from 20–100%. All samples were 50 mg / mL and were sonicated for 10 minutes before the experiment was performed at 15°C. (A) Images of the hydrogels formed at the indicated amplitudes. (B) Flow viscosity. The hydrodynamic data show that the viscosity of the gel decreases with more stress at all amplitudes tested, suggesting a shear-low viscosity material. (C) Time sweep. The hydrodynamic data show that storage > loss modulus at all concentrations at all amplitudes; i.e., it maintains the quality of the hydrogel. [Figure 12C] Figures 12A–12C show that acoustic hydrogels can be prepared by sonicating an ECM at a frequency of 20 kHz using amplitudes ranging from 20–100%. All samples were 50 mg / mL and were sonicated for 10 minutes before the experiment was performed at 15°C. (A) Images of the hydrogels formed at the indicated amplitudes. (B) Flow viscosity. The hydrodynamic data show that the viscosity of the gel decreases with more stress at all amplitudes tested, suggesting a shear-low viscosity material. (C) Time sweep. The hydrodynamic data show that storage > loss modulus at all concentrations at all amplitudes; i.e., it maintains the quality of the hydrogel. [Figure 13]Figure 13 shows that UBM acoustic hydrogel promotes the M2-like macrophage phenotype. Macrophages derived from mouse bone marrow were treated with 2 mg / ml UBM acoustic hydrogel for 24 hours, fixed, immunolabeled for strong indicators of the pro-inflammatory M1-like markers (iNos, TNFa) or remodeling-induced M2-like markers (izz1, arginase), and counterstained with DAPI. Treatment of cells with IFNγ and lipopolysaccharide (LPS) was used as a positive control for the M1-like phenotype, and IL-4 was used as a positive control for the M2-like phenotype. F4 / 80 staining was used as a positive control for macrophages. Cells were imaged at 200×. The data show that UBM acoustic hydrogel promoted the M2-like macrophage phenotype compared to the control. [Figure 14] Figures 14A - 14F show the sonication and temperature-induced gelation of fragmented ECM. (A) Demonstration of the immersion depth of the sonicator tip in a 50 ml conical tube. (B, C) Fragmented dermal ECM powder in 1X PBS before (B) and after (C) sonication. (D) After incubation at a temperature below 25°C, inversion of the tube showed that the solubilized ECM polymerized into a firm gel. (E) The polymerized gel could adapt to a 3D geometric structure. (F) The solubilized ECM can be transferred to a syringe (upper panel) and then cooled to a temperature below 25°C to give an injectable form of the gel (lower panel). [Figure 15A]Figures 15A–15D show the solubilization of collagen and sulfated glycosaminoglycans (sGAGs). (A) Concentration of solubilized collagen as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized collagen measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Concentration of sGAG as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized sGAG measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. (C) Concentration of solubilized collagen as a function of sonication time. Grinded dECM was sonicated for the indicated time at 100% amplitude. (D) Concentration of solubilized collagen as measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p < 0.05. Superscripts indicate paired comparisons. (D) Concentration of solubilized sGAG as a function of sonication time. Grinded dECM was sonicated at 100% amplitude for the indicated time. Concentration of solubilized sGAG was measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. [Figure 15B]Figures 15A–15D show the solubilization of collagen and sulfated glycosaminoglycans (sGAGs). (A) Concentration of solubilized collagen as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized collagen measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Concentration of sGAG as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized sGAG measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. (C) Concentration of solubilized collagen as a function of sonication time. Grinded dECM was sonicated for the indicated time at 100% amplitude. (D) Concentration of solubilized collagen as measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p < 0.05. Superscripts indicate paired comparisons. (D) Concentration of solubilized sGAG as a function of sonication time. Grinded dECM was sonicated at 100% amplitude for the indicated time. Concentration of solubilized sGAG was measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. [Figure 15C]Figures 15A–15D show the solubilization of collagen and sulfated glycosaminoglycans (sGAGs). (A) Concentration of solubilized collagen as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized collagen measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Concentration of sGAG as a function of sonication amplitude. Grinded dECM was sonicated for 300 seconds at the indicated amplitude. Concentration of solubilized sGAG measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. (C) Concentration of solubilized collagen as a function of sonication time. Grinded dECM was sonicated for the indicated time at 100% amplitude. (D) Concentration of solubilized collagen as measured using the SIRCOL® assay. Data are presented as mean ± sd of n=3 samples per group. * indicates p < 0.05. Superscripts indicate paired comparisons. (D) Concentration of solubilized sGAG as a function of sonication time. Grinded dECM was sonicated at 100% amplitude for the indicated time. Concentration of solubilized sGAG was measured using the BLYSCAN® assay. Data are presented as mean ± sd of n=3 samples per group. [Figure 15D]Figures 15A - 15D show the solubilization of collagen and sulfated glycosaminoglycan (sGAG). (A) Concentration of solubilized collagen as a function of sonication amplitude. The pulverized dECM was sonicated for 300 seconds at the indicated amplitudes. The concentration of solubilized collagen measured using the SIRCOL™ assay. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscript letters designate paired comparisons. (B) Concentration of sGAG as a function of sonication amplitude. The pulverized dECM was sonicated for 300 seconds at the indicated amplitudes. The concentration of solubilized sGAG measured using the BLYSCAN™ assay. Data are presented as mean ± s.d. for n = 3 samples per group. (C) Concentration of solubilized collagen as a function of sonication time. The pulverized dECM was sonicated at 100% amplitude for the indicated times. The concentration of solubilized collagen measured using the SIRCOL™ assay. Data are presented as mean ± s.d. for n = 3 samples per group. * represents p < 0.05. Superscript letters designate paired comparisons. (D) Concentration of solubilized sGAG as a function of sonication time. The pulverized dECM was sonicated at 100% amplitude for the indicated times. The concentration of solubilized sGAG was measured using the BLYSCAN™ assay. Data are presented as mean ± s.d. for n = 3 samples per group. [Figure 16A]Figures 16A–16C show the effects of temperature and sonication amplitude on the 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 seconds and then incubated at the indicated temperature to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Effect of sonication amplitude on gelation time. 25, 50, and 100 dECM were sonicated at the indicated amplitude for 300 seconds and then incubated at 4°C to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (C) Gelation assay to evaluate the effect of temperature on the gelation time of UBM, SIS, eECM, tECM, or LECM. Indicated tissue ECM at a concentration of 100 mg / ml was sonicated at 100% amplitude for 300 seconds, then incubated at 4°C or 25°C to induce gelation. Data are presented as mean ± sd for n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. [Figure 16B]Figures 16A–16C show the effects of temperature and sonication amplitude on the 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 seconds and then incubated at the indicated temperature to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Effect of sonication amplitude on gelation time. 25, 50, and 100 dECM were sonicated at the indicated amplitude for 300 seconds and then incubated at 4°C to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (C) Gelation assay to evaluate the effect of temperature on the gelation time of UBM, SIS, eECM, tECM, or LECM. Indicated tissue ECM at a concentration of 100 mg / ml was sonicated at 100% amplitude for 300 seconds, then incubated at 4°C or 25°C to induce gelation. Data are presented as mean ± sd for n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. [Figure 16C]Figures 16A–16C show the effects of temperature and sonication amplitude on the 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 seconds and then incubated at the indicated temperature to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (B) Effect of sonication amplitude on gelation time. 25, 50, and 100 dECM were sonicated at the indicated amplitude for 300 seconds and then incubated at 4°C to induce gelation. Data are presented as mean ± sd of n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. (C) Gelation assay to evaluate the effect of temperature on the gelation time of UBM, SIS, eECM, tECM, or LECM. Indicated tissue ECM at a concentration of 100 mg / ml was sonicated at 100% amplitude for 300 seconds, then incubated at 4°C or 25°C to induce gelation. Data are presented as mean ± sd for n=3 samples per group. * indicates p<0.05. Superscripts indicate paired comparisons. [Figure 17A] Figures 17A–17C show the viscoelastic characterization of ECM hydrogels prepared using ultrasonic cavitation. (A) Representative graphs of ECM hydrogel gelation kinetics at three temperature profiles for dECM and eECM. When the temperature decreased rapidly (37→4°C), the storage modulus (G') increased in an S-shape. When the temperature increased rapidly (4→37°C and 25→37°C), the hydrogel stiffness (G'') was maintained. (B) Average storage modulus at three temperature profiles (n=3, mean ±SD). (C) Average time to 50% gelation for the S-shaped temperature profile 37→4°C (n=3, mean ±SD). *p≦0.05, **p≦0.01. [Figure 17B]Figures 17A–17C show the viscoelastic characterization of ECM hydrogels prepared using ultrasonic cavitation. (A) Representative graphs of ECM hydrogel gelation kinetics at three temperature profiles for dECM and eECM. When the temperature decreased rapidly (37→4°C), the storage modulus (G') increased in an S-shape. When the temperature increased rapidly (4→37°C and 25→37°C), the hydrogel stiffness (G'') was maintained. (B) Average storage modulus at three temperature profiles (n=3, mean ±SD). (C) Average time to 50% gelation for the S-shaped temperature profile 37→4°C (n=3, mean ±SD). *p≦0.05, **p≦0.01. [Figure 17C] Figures 17A–17C show the viscoelastic characterization of ECM hydrogels prepared using ultrasonic cavitation. (A) Representative graphs of ECM hydrogel gelation kinetics at three temperature profiles for dECM and eECM. When the temperature decreased rapidly (37→4°C), the storage modulus (G') increased in an S-shape. When the temperature increased rapidly (4→37°C and 25→37°C), the hydrogel stiffness (G'') was maintained. (B) Average storage modulus at three temperature profiles (n=3, mean ±SD). (C) Average time to 50% gelation for the S-shaped temperature profile 37→4°C (n=3, mean ±SD). *p≦0.05, **p≦0.01. [Figure 18A-C]Figures 18A–18E show in vitro cellular responses. (A) 3T3 fibroblasts were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from UBM, SIS, or dermis, and cultured for 24 hours. Cellular metabolic activity was assessed using the VYBRANT® MTT cell proliferation assay kit. Data are presented as mean ± sd for n=3 samples per group. (B, C) Viability / death assay. Primary equine mesenchymal stem cells were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from dECM or UBM. Viability was assessed using a viability / death assay kit. Cells were imaged at 200× (B), and percentage viable and dead cells were quantified using a cell profiler (C). Data are presented as mean ± sd for n=3 samples per group. (D) Mouse bone marrow-derived macrophages were either untreated (control) or treated for 24 hours with one of the following test products: IFNγ+LPS, IL-4, dECM hydrogel, or eECM hydrogel. Cells were immunolabeled with F4 / 80 (macrophage marker), iNOS (M1-like marker), or Fizz1 (M2-like marker). Cells were imaged at 200×. (E) Quantification of F4 / 80, iNOS, and Fizz1 immunolabeling. Data are presented as mean ± sd for n=3 per group. [Figure 18D]Figures 18A–18E show in vitro cellular responses. (A) 3T3 fibroblasts were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from UBM, SIS, or dermis, and cultured for 24 hours. Cellular metabolic activity was assessed using the VYBRANT® MTT cell proliferation assay kit. Data are presented as mean ± sd for n=3 samples per group. (B, C) Viability / death assay. Primary equine mesenchymal stem cells were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from dECM or UBM. Viability was assessed using a viability / death assay kit. Cells were imaged at 200× (B), and percentage viable and dead cells were quantified using a cell profiler (C). Data are presented as mean ± sd for n=3 samples per group. (D) Mouse bone marrow-derived macrophages were either untreated (control) or treated for 24 hours with one of the following test products: IFNγ+LPS, IL-4, dECM hydrogel, or eECM hydrogel. Cells were immunolabeled with F4 / 80 (macrophage marker), iNOS (M1-like marker), or Fizz1 (M2-like marker). Cells were imaged at 200×. (E) Quantification of F4 / 80, iNOS, and Fizz1 immunolabeling. Data are presented as mean ± sd for n=3 per group. [Figure 18E]Figures 18A–18E show in vitro cellular responses. (A) 3T3 fibroblasts were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from UBM, SIS, or dermis, and cultured for 24 hours. Cellular metabolic activity was assessed using the VYBRANT® MTT cell proliferation assay kit. Data are presented as mean ± sd for n=3 samples per group. (B, C) Viability / death assay. Primary equine mesenchymal stem cells were seeded on petri dishes coated with control (uncoated) or ECM hydrogel prepared from dECM or UBM. Viability was assessed using a viability / death assay kit. Cells were imaged at 200× (B), and percentage viable and dead cells were quantified using a cell profiler (C). Data are presented as mean ± sd for n=3 samples per group. (D) Mouse bone marrow-derived macrophages were either untreated (control) or treated for 24 hours with one of the following test products: IFNγ+LPS, IL-4, dECM hydrogel, or eECM hydrogel. Cells were immunolabeled with F4 / 80 (macrophage marker), iNOS (M1-like marker), or Fizz1 (M2-like marker). Cells were imaged at 200×. (E) Quantification of F4 / 80, iNOS, and Fizz1 immunolabeling. Data are presented as mean ± sd for n=3 per group. [Figure 19] Figures 19A-19C show acoustic hydrogels as submucosal fluid cushions. (A) Acoustic extracellular matrix (ECM) hydrogels (100 mg / mL) were prepared from dermal ECM (dECM) and esophageal mucosal ECM (eECM) and used ex vivo as submucosal fluid cushions. The effect of 20 kGy gamma irradiation (γ) on the height of the acoustic ECM hydrogel fluid cushion was evaluated. Clinical standards Eleview and PBS were used as controls. The height of the fluid cushion was measured over time after injection of 2 mL of the test sample into the porcine esophagus. Values ​​are expressed as mean ± SD (n=3). (B) Acoustic hydrogel samples can be injected using a 16G syringe. (C) Representative photograph of the height of the test sample fluid cushion after 75 minutes. [Figure 20A]Figures 20A–20B show acoustic hydrogel formation. Hydrogel "stiffness" over time was measured for gamma-ray irradiation (20 kGy) and non-sterile control acoustic hydrogel (dermal ECM 100 mg / mL). Storage modulus ("stiffness") (G') and loss modulus (G") were measured by applying a small 0.5% vibrational tension to the sample. Three temperature profiles were tested: temperature was rapidly increased from the initial storage temperature to the final temperature: 4 to 37°C, 25 to 37°C, or 37 to 4°C. (A) A representative graph of the time sweep is shown (B). The average storage and loss moduli averaged over the last 5 minutes of the test are shown. [Figure 20B] Figures 20A–20B show acoustic hydrogel formation. Hydrogel "stiffness" over time was measured for gamma-ray irradiation (20 kGy) and non-sterile control acoustic hydrogel (dermal ECM 100 mg / mL). Storage modulus ("stiffness") (G') and loss modulus (G") were measured by applying a small 0.5% vibrational tension to the sample. Three temperature profiles were tested: temperature was rapidly increased from the initial storage temperature to the final temperature: 4 to 37°C, 25 to 37°C, or 37 to 4°C. (A) A representative graph of the time sweep is shown (B). The average storage and loss moduli averaged over the last 5 minutes of the test are shown. [Modes for carrying out the invention]

[0009] Detailed description of several embodiments To promote the repair and reconstruction of various tissues, ECM hydrogels are used as substrates for 3D organoid cultures and in numerous preclinical and clinical applications. Previously, ECM hydrogel materials were prepared using laborious methods focusing on the enzymatic digestion of ECM with acidic proteases in acidic solutions; or by the use of chaotropic extraction buffers and dialysis procedures, which can affect the innate protein structure and function. Herein, a method for preparing hydrogels from an ECM bioskeleton using sonic cavitation is disclosed. By controlling the temperature, the solubilized ECM can be induced to rapidly self-assemble into a gel, and the material properties of the gel can be modified by adjusting the ECM concentration and sonication parameters. The ECM bioskeleton can be successfully solubilized using ultrasound without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell proliferation. These hydrogels can be used in numerous applications and can be sterilized at the end by gamma irradiation.

[0010] To produce the disclosed ECM hydrogel, the sonication technique can be applied to many tissue-specific ECMs, including, but not limited to, the dermis, bladder matrix (UBM), and small intestinal submucosa (SIS). It can also be used with commercially available ECM formulations. In some embodiments, this approach involves resuspending the pulverized ECM in a liquid, such as a buffer solution, such as a neutral buffered saline solution, followed by solubilization of the ECM using sonication. In some embodiments, the buffered saline solution has a molar osmotic concentration 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. Gelation time and ECM gel properties can be adjusted by adjusting the ECM concentration, sonication amplitude, and duration. In some embodiments, the acoustic ECM hydrogel does not contain exogenous proteases or inactivated exogenous proteases, such as exogenous pepsin, trypsin, or hyaluronidase, or inactive forms of exogenous pepsin, trypsin, or hyaluronidase.

[0011] Upon polymerization, these ECM hydrogels are stable at room temperature and can adapt to customizable 3D geometric structures. ECM hydrogels produced by sonication ("acoustic ECM hydrogels") can be processed into a solid skeleton by freeze-drying procedures that increase porosity while maintaining the overall 3D geometric structure. 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, for example, to increase hemostasis.

[0012] Relatively small progress has been made in the large-scale production of ECM hydrogels (Brown et al., 2012, see above). The disclosed method is useful for large-scale production in several embodiments. In some embodiments, the concentration range of the ECM hydrogel can be extended from 2–20 mg / ml (limit of the enzymatic method) to 25–100 mg / ml using an ultrasonic cavitation method, which allows for fine-tuning of the viscoelastic properties of the ECM hydrogel for specific clinical applications. In other embodiments, the processing time is drastically reduced from 48–72 hours to the order of minutes. In further embodiments, the ECM hydrogel can adapt to a customizable 3D geometric structure, supporting the incorporation of cells or therapeutic agents for in vitro and in vivo applications.

[0013] term Unless otherwise specified, technical terms will be used according to their conventional usage. The definitions of general terms in molecular biology are found in Krebs et al., published by Jones & Bartlett Publishers. This can be found in (Eds.), Lewin's Genes XII, 2017; and in Volume 16 of Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, 2008, published by Wiley-VCH; and other similar references.

[0014] To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0015] Acid proteases are enzymes that cleave peptide bonds, and whose activity in cleaving peptide bonds increases at acidic pH. For example, acid proteases can include pepsin and trypsin, but are not limited to these.

[0016] Antibiotics: Compounds or substances that kill bacteria, fungi, or any other microorganisms, or significantly slow down their rate of growth. "Antibacterial agents" are compounds or substances that kill bacteria or significantly slow down their rate of growth.

[0017] Antibacterial antibiotics are generally classified based on their mechanism of action, chemical structure, or activity spectrum. Most target bacterial function or growth processes. Those that target the bacterial cell wall (e.g., penicillins and cephalosporins) or cell membrane (e.g., polymyxins), or that interfere with essential bacterial enzymes (e.g., quinolones and sulfonamides) are bactericidal. Those that target protein synthesis (e.g., aminoglycosides, macrolides, and tetracyclines) are generally bacteriostatic. Further classification is based on their target specificity.

[0018] "Narrow-spectrum" antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria. "Broad-spectrum" antibiotics affect several different types of bacteria. Antibacterial agents also include cyclic lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), and oxazolidinones (e.g., linezolid).

[0019] Topical antibiotics are antibiotics applied to the skin or other body surfaces such as the eyes. Topical antibiotics are often 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), pseudomonic acid (e.g., mupirocin), aminoglycosides (e.g., neomycin), or quinolones (e.g., ciprofloxacin or ofloxacin), or nitroimidazoles (e.g., metronidazole). It contains, or a combination of drugs (such as bacitracin / polymyxin or neomycin / polymyxin B / bacitracin).

[0020] Biocompatibility: Any material that, when implanted in a mammalian subject, does not cause a harmful response in the subject. When introduced into an individual, a biocompatible material can perform its intended function, is neither toxic nor harmful to the individual, and does not induce immunological rejection of the material in the subject.

[0021] Biosynthetic skeleton: A biocompatible skeleton, usually a solid support or gel. Biosynthetic skeletons are composed of naturally occurring materials. Biosynthetic skeletons are composed of materials that do not exist naturally and materials that do exist naturally.

[0022] Centrifugal separation is the process by which centrifugal force is applied to a mixture, thereby causing the denser components of the mixture to move away from the axis of the centrifuge compared to other less dense components in the mixture. The force applied to the mixture is a function of the velocity and radius of rotation of the centrifuge rotor. In most applications, the rotational force results in a precipitate (pellet) that accumulates at the bottom of the centrifuge tube, where the remaining solution is precisely called the "supernatant" or "clear liquid." In other similar applications, density-based separation or "density gradient centrifugation" techniques are used to isolate specific species from mixtures containing both denser and less dense components than the desired component.

[0023] During the annular motion of a centrifugal rotor, the applied force is the product of the radius of rotation and the angular velocity, where the force is traditionally expressed as an acceleration compared to the standard acceleration "g" due to gravity on the Earth's surface. The applied centrifugal force is called the "relative centrifugal force" (RCF) and is expressed as a multiple of "g".

[0024] Grinding (grinding and pulverizing): A process of reducing larger particles to smaller particles, including, but not limited to, grinding, blending, crushing, slicing, fine grinding, cutting, and shredding. ECM can be ground into any form, without limitation, including hydrated, freeze-dried, air-dried, freeze-dried, powder, and sheet forms.

[0025] Contact: Placement into direct physical contact, whether in solid or liquid form.

[0026] Cytokines: The term "cytokines" is used as a general term for a diverse group of soluble proteins and peptides that act as humoral regulators at concentrations ranging from nano to picomoles, modulating the functional activity of individual cells and tissues under normal or pathological conditions. These proteins also directly mediate intercellular interactions and regulate processes occurring in the extracellular environment. Examples of cytokines include, but are not limited to, tumor necrosis factor-α, interleukin (IL)-6, IL-10, IL-12, transforming growth factors, and interferon-γ.

[0027] Diagnosis: The process of identifying a disease based on its signs, symptoms, and the results of various tests. The conclusion reached through this process is also called a "diagnosis." Common forms of testing include blood tests, medical imaging, and biopsies.

[0028] Extracellular matrix (ECM): The natural, non-cytoskeletonized tissue for cell proliferation. Natural ECM (ECM found in mammals and multicellular organisms such as humans, without limitation) is a complex mixture of structural and non-structural biomolecules, including, but not limited to, collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial substances, chemoattractants, cytokines, and growth factors. In mammals, ECM often contains approximately 90% collagen in its various forms. The composition and structure of ECM vary depending on the tissue source. For example, the submucosa of the small intestine (SIS), bladder matrix (UBM), esophagus (E), and liver stromal ECM each have different overall structures and compositions due to the specific cellular niches required by each tissue. The intact "extracellular matrix" and "intact ECM" are extracellular matrices that retain the activity of their structural and non-structural biomolecules, including, but not limited to, collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobial substances, chemoattractants, cytokines, and growth factors.

[0029] The structure and / or activity of biomolecules within the ECM can be altered or removed chemically or mechanically, for example, by crosslinking and / or by dialysis of the ECM. An intact ECM is essentially enzymatically undigested, uncrosslinked, and / or undialysisd, meaning that the ECM has not been subjected to digestion, dialysis, and / or crosslinking processes, or conditions other than those that occur naturally during the storage and handling of the ECM prior to solubilization. Therefore, an ECM that is substantially crosslinked and / or dialyzed (crosslinked and / or dialyzed in any manner other than in an insignificant manner that does not substantially affect the gelation and functional characteristics of the ECM in its use as described herein) is not considered “intact.” “Cellless” refers to an ECM produced from a donor tissue that has been treated to remove cells so that the ECM remains. Cell-free tissue is used to produce the ECM hydrogel.

[0030] A gel is a state of matter between liquid and solid, generally defined as a cross-linked polymer network that swells in a liquid medium. Typically, a gel is a two-phase colloidal dispersion containing both solid and liquid, with the amount of solid being greater than that of a two-phase colloidal dispersion called a "sol." Thus, a "gel" possesses some of the properties of a liquid (i.e., its shape is elastic and deformable) and some of the properties of a solid (e.g., its shape is discrete enough to maintain three dimensions on a two-dimensional surface). "Gelization time," also called "gel time," refers to the time it takes for a composition to become non-flowing under mild stress.

[0031] Gelation: Formation of a gel from a sol.

[0032] Hemostasis: To stop or halt bleeding.

[0033] Hydrogels: Networks of hydrophilic polymer chains, sometimes found as colloidal gels with water as the dispersion medium. Hydrogels are highly absorbent natural or synthetic polymer networks. Hydrogels also possess a degree of flexibility similar to that of natural tissues. "Acoustic" hydrogels, such as acoustic ECM hydrogels, are produced using ultrasonic energy. The characteristics of these hydrogels are disclosed herein. In the case of hydrogels, the storage modulus (G') is generally about an order of magnitude larger than the loss modulus (G'').

[0034] Isolated: “Isolated” biological components (such as the extracellular matrix) are substantially separated, produced separately, or purified from other biological components, cells or organisms in which the components naturally occur, i.e., living cells, other chromosomes and extrachromosomal DNA and RNA, and proteins. Therefore, “isolated” nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. This term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemosynthesized nucleic acids. Isolated ECM is isolated from the cells that produce the ECM.

[0035] Isotonic buffer solution: A solution buffered between pH 7.2 and 7.8, with an equilibrium concentration of salts to promote an isotonic environment.

[0036] Macrophages are a type of white blood cell that phagocytoses and breaks down cell fragments, foreign substances, microorganisms, and cancer cells. In addition to their role in phagocytosis, these cells play a crucial role in both innate and adaptive immunity, as well as in development, tissue maintenance and repair, and in their ability to mobilize and influence other cells, including immune cells such as lymphocytes. Macrophages can exist in many phenotypes, including those called M1 and M2, also known as "M1-like" and "M2-like." Macrophages that primarily exert pro-inflammatory functions are called M1 macrophages (CD86+ / CD68+), while macrophages that reduce inflammation and promote and regulate tissue repair are called M2 macrophages (CD206+ / CD68+). Markers that identify the various phenotypes of macrophages differ among species. It should be noted that macrophage phenotypes are represented by a spectrum ranging between the M1 and M2 extremes.

[0037] Mammals: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

[0038] Preventing or treating a disease: “Preventing” a disease refers to preventing the partial or complete development of a disease in a person known to have a predisposition to a disease, such as cancer. Examples of people with a known predisposition include those with a family history of breast cancer or those exposed to factors that predispose the subject to a condition such as melanoma. “Treatment” refers to therapeutic interventions that improve the signs or symptoms of a disease or pathological condition after it has begun to develop. In some embodiments, treatment refers to reducing the size of a tumor, reducing the number and / or size of metastases, or alleviating the symptoms of a tumor.

[0039] Therapeutic agents: When used in a general sense, this includes procedural agents, prophylactic agents, and replacement agents. "Procedure" or "to treat" means providing a patient with a substance such as acoustic ECM hydrogel in an amount sufficient to clearly affect a biological parameter, for example, to increase hemostasis.

[0040] Therapeutic Effective Dose: The “therapeutic effective dose” of a composition such as acoustic ECM hydrogel means the amount that, when administered to a patient, is effective in providing therapeutic benefits, such as improvement of symptoms, reduction of disease progression, or in causing disease regression. The amount of acoustic ECM hydrogel is sufficient to achieve the desired effect in the subject being treated. The therapeutic effective dose can be administered systemically or topically, for example, to a wound. Furthermore, the effective dose of acoustic ECM hydrogel can be administered in a single dose or in several doses over time. However, the effective dose depends on the formulation being administered, the subject being treated, the severity and type of disease, and the mode of administration of the compound. Acoustic ECM hydrogels useful in the manner disclosed herein have equal applications in medical and veterinary settings. Therefore, the general terms “subject” or “patient” are understood to include all animals, including, but not limited to, human or veterinary subjects, such as other primates, dogs, cats, horses, and cattle.

[0041] Thermoreversible hydrogels are hydrogels formed by the entanglement of polymer chains, in which viscosity changes at a temperature characteristic of gelation. The disclosed acoustic ECM hydrogel is a thermoreversible hydrogel that exhibits gelation (transition from sol to gel) upon cooling.

[0042] Topical application: Topically applied drugs are applied only to a specific area and not to the entire body. In certain cases, the composition is applied to the skin or eye in an area where hemostasis is desired. For example, a pharmaceutical composition can be applied 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 an abrasion or surgical incision of the skin or cornea.

[0043] Ultrasonic treatment: A process that involves exposure to ultrasound at frequencies higher than 20 kHz.

[0044] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise. Similarly, the word “or” includes “and” unless the context clearly indicates otherwise. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for description purposes only. “Approximately” indicates within 5% of the listed value. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but suitable methods and materials are listed below. The term “comprises” means “includes.” All publications, patent applications, patents, and other references pointed out herein are incorporated entirely by reference. In case of conflict, this specification, including definitions of terms, shall prevail. Furthermore, materials, methods, and examples are illustrative and not limiting.

[0045] Extracellular matrix (ECM) Any type of extracellular matrix tissue can be used to generate hydrogels (related to ECM, U.S. Patent Nos. 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5,762,966; 5, See Nos. 866,414; 6,099,567; 6,485,723; 6,576,265; 6,579,538; 6,696,270; 6,783,776; 6,793,939; 6,849,273; 6,852,339; 6,861,074; 6,887,495; 6,890,562; 6,890,563; 6,890,564; and 6,893,666). In certain embodiments, the ECM is isolated from vertebrates, for example, from warm-blooded mammalian vertebrates, including, but not limited to, humans, monkeys, horses, pigs, cattle, and sheep. In specific non-limiting examples, the ECM is a pig or a human.

[0046] ECM can be derived from any organ or tissue, including but not limited to the bladder, intestines (such as the small or large intestine), heart, kidneys, uterus, brain, blood vessels, lungs, bones and muscles, pancreas, stomach, spleen, adipose tissue, liver, esophagus, and dermis. ECM can be obtained from cell culture. In one embodiment, ECM is isolated from the bladder. In another embodiment, ECM is derived from the esophagus. In yet another embodiment, ECM is derived from the dermis. ECM may or may not include a basement membrane portion of the ECM. In certain embodiments, ECM includes at least a portion of the basement membrane. To produce ECM, tissue can be decellularized, for example, to remove cells and cellular material from the source tissue or organ. It is desirable to use decellularized material to inhibit an immune response, such as when implanting ECM into a subject as a component of a hydrogel disclosed herein. Removal of cellular material, such as when forming a hydrogel using ECM, inhibits such an immune response.

[0047] U.S. Patent No. 8,361,503 (incorporated herein by reference) discloses the preparation of a bladder ECM, for example, a pig bladder ECM is prepared by scraping the bladder tissue to remove the outer layers, including both the serosal and muscular layers, using a longitudinal wiping motion with a scalpel handle and wet gauze. After abduction of the tissue segments, the luminal portion of the mucosa is separated from the underlying tissue using the same wiping motion. In some embodiments, perforation of the submucosa is prevented. After removal of these tissues, the resulting ECM consists mainly of the submucosa.

[0048] The generation of hydrogels from dermal ECM is incorporated herein by reference by Wolf et al. Disclosed in al., Biomaterials 33: 7028-7038, 2012. The life of the ECM from esophageal tissue. The results are disclosed, for example, in Badylak et al. J Pediatr Surg. 35(7):1097-103, 2000 and Badylak et al., J Surg Res. 2005 September; 128(1):87-97, 2005. Both are incorporated herein by reference. U.S. Patent No. 6,893,666, incorporated herein by reference, discloses the production of ECM from the bladder, skin, esophagus, and small intestine. ECM can be produced from any of these tissues.

[0049] Commercially available ECM formulations can also be used. In one embodiment, the ECM is derived from the submucosa of the small intestine or SIS. Commercially available formulations include, but are not limited to, SURGISIS®, SURGISIS-ES®, STRATASIS®, and STRATASIS-ES® (Cook Urological Inc.; Indianapolis, Ind.), and GRAFTPATCH® (Organogenesis Inc.; Canton Mass.). In another embodiment, the ECM is derived from the dermis. Commercially available formulations include, but are not limited to, PELVICOL® (marketed as PERMACOL® in Europe; Bard, Covington, Ga.), REPLIFORM® (Microvasive; Boston, Mass.), and ALLODERM® (LifeCell; Branchburg, NJ). In yet another embodiment, the ECM is derived from the bladder. Commercially available formulations include, but are not limited to, UBM (Acell Corporation; Jessup, Md.).

[0050] Tissue for the preparation of ECM can be harvested in various ways, and once harvested, different parts of the harvested tissue can be used. ECM has also been prepared from the esophagus and small intestine, for example, Keane et al., Tissue Eng., which is incorporated herein by reference. See Part A, 21(17-18): 2293-2300, 2015. Esophageal ECM can be prepared by mechanically separating the mucosa and submucosa from the muscularis exomucosa, digesting the mucosa with a trypsin-containing buffer, and subsequently exposing it to sucrose, TRITON-X100®, deoxycholic acid, peracetic acid, and DNase. Small intestinal submucosa (SIS) can be prepared by mechanically removing the superficial layers of the mucosa, serosal layer, and muscularis exomucosa from an intact small intestine, leaving intact submucosa, muscularis mucosa, and basal compacta. SIS is then treated with peracetic acid. An exemplary protocol is provided in Keane et al. Dermal hydrogels are disclosed, for example, in Wolf et al, J Biomed Mater Res A. 2013. 35(25):6838-49. PMID: 23873846. PMCID: 3808505, which are incorporated herein by reference. It can be generated in such a way.

[0051] In one embodiment, the extracellular matrix (ECM) is isolated from a collected pig bladder to prepare the bladder matrix (UBM). Excess connective tissue and residual urine are removed from the bladder. The serosal layer, muscular layer, submucosa, and most of the muscularis mucosa can be removed by mechanical scraping or by a combination of enzymatic treatment, hydration, and scraping. Mechanical removal of these tissues can be achieved by scraping with longitudinal wiping motions to remove the outer layers of the mucosa (epithelial layer) (particularly the smooth muscle layer on the anti-luminal side) and even the luminal portion. Mechanical removal of these tissues can be achieved, for example, by removing the mesenteric tissue with Adson-Brown forceps and Metzenbaum scissors, and by wiping away the muscular and submucosal layers using longitudinal wiping motions with a scalpel handle wrapped in wet gauze or other firm object. Epithelial cells of the mucosa can also be dissociated by immersing the tissue in a deepithelializing solution, for example, but not limited to, hypertonic saline. The resulting UBM contains the mucosal basement membrane and adjacent lamina propria, which is further treated with peracetic acid, freeze-dried, and powdered. See U.S. Patent No. 8,361,503 incorporated herein by reference.

[0052] Dermal sections may be used for the preparation of ECM hydrogels; see PCT application number 2015 / 15164728 incorporated herein by reference. In a specific, non-limiting example, the dermis can be decellularized with 0.25% trypsin / 1% TRITON-X(registered trademark)-100 (i.e., without SDS) by stirring and shaking at 300 RPM in the following solutions at room temperature: 0.25% trypsin for 6 hours, 1×; deionized water, 15 minutes, 3×; 70% ethanol, 10 to 12 hours, 1×; 3% H2O2, 15 minutes, 1×; deionized water, 15 minutes, 2×; 1% TRITON-X(registered trademark)-100 in 0.26% EDTA / 0.69% Tris, 6 hours, 1×, then overnight, 1×; deionized water, 15 minutes, 3×; 0.1% peracetic acid / 4% ethanol, 2 hours, 1×; PBS, 15 minutes, 2×; finally deionized water, 15 minutes, 2×. The dermal sheets are then freeze-dried and subsequently reduced to particle form using a Waring blender and a Wiley mill with a #20 mesh screen.

[0053] In some embodiments, epithelial cells can first be aschone by immersing the tissue in a deepithelializing solution, such as hypertonic saline, for a period ranging from 10 minutes to 4 hours, without limitation. Exposure to hypertonic saline effectively removes epithelial cells from the underlying basement membrane. The tissue remaining after the initial aschoneting procedure includes the epithelial basement membrane and the tissue layer antiluminal to the epithelial basement membrane. This tissue is then subjected to further processing to remove most of the antiluminal tissue rather than the epithelial basement membrane. The outer serosa, adventitia, smooth muscle tissue, submucosa, and most of the muscularis mucosa are removed from the remaining deepithelialized tissue by mechanical scraping or by a combination of enzymatic treatment, hydration, and scraping.

[0054] ECM can be sterilized by any number of standard techniques, including, but not limited to, exposure to peracetic acid, low-dose gamma-ray irradiation, gas plasma sterilization, ethylene oxide treatment, or electron beam treatment. More generally, sterilization of ECM is achieved by immersion for 2 hours in 0.1% (v / v) peracetic acid, 4% (v / v) ethanol, and 95.9% (v / v) sterile water. Peracetic acid residue is removed by two 15-minute washes with PBS (pH=7.4) and two 15-minute washes with sterile water. ECM materials can be sterilized by propylene oxide or ethylene oxide treatment, gamma-ray irradiation (0.05 to 4 mRad), gas plasma sterilization, peracetic acid sterilization, or electron beam treatment. ECM can also be sterilized by treatment with glutaraldehyde, which causes crosslinking of the protein material, but this treatment substantially modifies the material so that it is gradually reabsorbed or not reabsorbed at all, stimulating a different type of host remodeling that closely resembles scar tissue formation or encapsulation rather than constructive remodeling. Crosslinking of the protein material can also be induced by carbodiimide or deheated water or photo-oxidation methods. As disclosed in U.S. Patent No. 8,361,503, ECM is disinfected by immersion for 2 hours in 0.1% (v / v) peracetic acid (a), 4% (v / v) ethanol and 96% (v / v) sterile water. The ECM material is then washed twice for 15 minutes with PBS (pH=7.4) and twice for 15 minutes with deionized water.

[0055] Generally, after isolation of the target tissue, decellularization is carried out by various methods, for example, by exposure to hypertonic saline, peracetic acid, TRITON-X®, or other cleaning agents, without limitation. Sterilization and decellularization may be performed simultaneously. For example, sterilization with peracetic acid as described above may also be used for decellularization, without limitation. The ECM can then be dried by freeze-drying or air-drying. The dried ECM can be pulverized by methods including, without limitation, tearing, grinding, cutting, grinding, and shearing. The pulverized ECM can be further processed into a powder by methods such as grinding or pulverizing in a frozen or freeze-dried state, without limitation.

[0056] Mammalian ECMs are also commercially available. These include AVITENE®, MICROMATRIX®, and XENMATRIX®.

[0057] Acoustic ECM hydrogels and their preparations In some embodiments, the ground ECM, such as mammalian ECM, is diluted in a liquid. The ECM may or may not be freeze-dried before grinding. The ECM can be ground, for example, by grinding, shredding, or cutting. The ground ECM should have fragments in the range of about 10 μm to about 5000 μm, about 10 μm to about 4000 μm, about 10 μm to about 3000 μm, about 10 μm to about 2000 μm, about 10 μm to about 1000 μm, about 10 μm to about 500 μm, about 30 μm to about 300 μm, about 40 μm to about 400 μm, about 25 μm to about 500 μm, about 50 μm to about 500 μm, about 100 μm to about 300 μm, about 10 μm to about 50 μm, or about 10 μm to about 100 μm. In one embodiment, the ECM is provided in fragments ranging from about 10 μm to about 1000 μm. In another preferred embodiment, the ECM is provided in fragments ranging from about 10 μm to about 2000 μm. In one non-limiting example, the fragments are in the range of about 30 μm to about 300 μm. The liquid may be a buffer with a neutral pH, for example, pH of about 7.0 to about 7.6, for example, about 7.1 to about 7.5, for example, about 7.2 to about 7.4, for example, about 7.0 to 7.2, for example, about 7.0 to 7.4, for example, 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, for example, phosphate-buffered saline (PBS) or Tris-buffered saline, without limitation. In some embodiments, the buffered saline solution has a molar osmotic concentration of about 290 mOsm / L. The liquid may be water. In some embodiments, an isotonic buffer containing, without limitation, phosphate-buffered saline (PBS) may be used to bring the solution to a target pH or to help maintain the pH and ionic strength of the gel at target levels such as physiological pH and ionic conditions. This forms a liquid ECM solution.

[0058] The methods disclosed generally do not involve the use of acid proteases, including pepsin, trypsin, or hyaluronidase. See PCT application number WO2015 / 164728, incorporated herein by reference. Generally, in these methods, the solubilized ECM in liquid does not come into contact with the acid protease.

[0059] In some embodiments, ECM is used in liquids at concentrations exceeding approximately 25 mg / ml. ECM can be used in liquids such as buffers at concentrations ranging from approximately 25 mg / ml to approximately 600 mg / ml. Suitable concentrations also include approximately 25 mg / ml to approximately 300 mg / ml, approximately 25 mg / ml to approximately 200 mg / ml, and approximately 25 mg / ml to approximately 150 mg / ml. ECM can be used in liquids such as buffers at concentrations ranging from approximately 50 mg / ml to approximately 600 mg / ml. Suitable concentrations also include approximately 50 mg / ml to approximately 300 mg / ml, approximately 50 mg / ml to approximately 200 mg / ml, and approximately 50 mg / ml to approximately 150 mg / ml. Suitable concentrations include approximately 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 approximately 25 mg / ml, 100 mg / ml, and 150 mg / ml. In one non-limiting example, the ECM is in a liquid at a concentration of approximately 25 mg / ml to approximately 150 mg / ml. In another non-limiting example, the ECM is in a liquid at a concentration of 100 mg / ml.

[0060] ECM in liquids such as buffered saline solutions is treated with ultrasonic frequencies. In one embodiment, the ultrasonic frequencies are approximately 20 kHz to approximately 100 kHz. ECM in liquids can be treated with ultrasonic frequencies of approximately 20 kHz to approximately 30 kHz, approximately 20 kHz to approximately 40 kHz, approximately 20 kHz to approximately 50 kHz, approximately 20 kHz to approximately 60 kHz, approximately 20 kHz to approximately 70 kHz, approximately 20 kHz to approximately 80 kHz, or approximately 20 kHz to approximately 90 kHz. ECM in liquids can be treated with ultrasonic frequencies of approximately 20 kHz, 30 kHz, 40 kHz, 50 kHz, 60 kHz, 70 kHz, 80 kHz, 90 kHz, or 100 kHz. In one non-limiting example, ECM in liquids can be treated with ultrasonic frequencies of approximately 20 kHz.

[0061] ECM in a liquid such as a buffered saline solution is treated with ultrasound for at least 20 seconds, for example, at least 30 seconds. ECM in a liquid such as a buffered saline solution is treated with ultrasound for at least 60 seconds. In some embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 1 hour. In further embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 30 minutes. In further embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 30 minutes. In further embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 15 minutes. In further embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 15 minutes. In some embodiments, ECM in a liquid is treated with ultrasound for at least 60 seconds to about 10 minutes. In some embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 10 minutes. In some embodiments, ECM in a liquid is treated with ultrasound for at least 30 seconds to about 5 minutes. In some embodiments, the ECM in liquid is treated with ultrasound for at least 30 seconds to about 5 minutes. The ECM in 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 liquid is treated with pulsed ultrasound for the total time listed herein. Therefore, in some embodiments, the ECM in a liquid such as a buffered saline solution is treated with pulses of, for example, a length of at least about 30 seconds, for example, about 30, about 40, or about 60 seconds. The ECM in a liquid such as a buffered saline solution can be treated with ultrasound 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, such that the total treatment time is 60 seconds to 1 hour, or any of the listed total times.ECM in liquids such as saline solution can be applied for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 seconds. ECM in liquids such as saline solution can be applied for at least 30 seconds. Generally, when multiple treatments are used, they occur within less than one hour. An exemplary method is a 30-second pulse of ultrasound followed by 30-45 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, for example, six 30-second pulses of ultrasound at approximately 20 kHz, followed by a 45-second pause, repeated six times, for a total of three minutes of ultrasound treatment.

[0062] Ultrasound can have an amplitude of approximately 20 μm to approximately 320 μm. Generally, the amplitude is measured from the center of the probe used to generate the ultrasound. The amplitude of the vibrating surface of the probe is the distance between the fully extended and fully retracted positions of the probe and is measured in microns (μm). In some embodiments, the amplitude is approximately 30 μm to approximately 200 μm. In further embodiments, the amplitude is approximately 36 μm to approximately 180 μm. The amplitude may be approximately 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 150, 160, 70, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 μm. In some embodiments, the amplitude is approximately 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm, 150-160 μm, and 160-170 μm. The amplitude may be 170-180 μm, 180-190 μm, 190-200 μm, 200-210 μm, 210-220 μm, 220-230 μm, 230-240 μm, 240-250 μm, 250-260 μm, 260-270 μm, 270-280 μm, 280-290 μm, or 290-300 μm. In one specific non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm. In a further non-limiting example, the ultrasound has a frequency of approximately 20 kHz and an amplitude of approximately 36 μm to approximately 180 μm, and the treatment is for a total of approximately 1, 2, 3, 4, or 5 minutes, for example, approximately 3 minutes. The ultrasonic treatment may 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 ultrasonic treatment may be from about 30 seconds to about 5 minutes. The ultrasonic treatment may be, for example, between about 1 and about 5 minutes. The ultrasonic treatment may be from about 1 to about 10 minutes. The ultrasonic treatment may be, for example, between 1 and about 20 minutes. In further embodiments, the ultrasonic treatment may be 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 ultrasonic treatment may be for at least 30 seconds.In other embodiments, the ultrasonic treatment may last from about 10 minutes to about 24 hours, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the ultrasonic treatment may last up to 48 hours.

[0063] In some embodiments, the ECM in liquid is ultrasonically treated at temperatures ranging from about 30°C to about 43°C. In one embodiment, the ECM in liquid is ultrasonically treated at temperatures ranging from about 35°C to about 40°C. In another embodiment, the ECM in liquid is ultrasonically treated at temperatures ranging from about 36°C to about 38°C. In yet another embodiment, the ECM in liquid is ultrasonically treated at temperatures ranging from about 37°C or higher, for example, from about 37°C to about 55°C, for example, from about 37°C to about 50°C, for example, from about 37°C to about 45°C, for example, from about 37°C to about 40°C. The ECM in liquid is ultrasonically treated at temperatures ranging from about 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55°C. In further embodiments, the ECM in liquid is ultrasonically treated at a temperature higher than about 38°C, for example, about 38°C to about 50°C, for example, about 38°C to about 45°C, for example, about 38°C to about 40°C.

[0064] Ultrasonic treatment generates an acoustic ECM hydrogel. Acoustic ECM hydrogels generally undergo a phase transition from sol to gel at around 37°C, and thus transition to a liquid phase at temperatures above 37°C and to a gel phase below 37°C. At 37°C, the acoustic ECM hydrogel is sufficiently viscous to resemble a gel, but as the temperature rises above 37°C, the gel transitions to a sol. The acoustic ECM hydrogel forms a gel upon a decrease in temperature below 37°C (sol-to-gel transition). Therefore, in some embodiments, after sonication, the acoustic ECM hydrogel is cooled to a temperature below 37°C, for example, from about 4°C to about 36°C. The acoustic ECM hydrogel can generally be cooled to room temperature, which is about 25°C. In some embodiments, the acoustic ECM hydrogel is cooled to about 15°C to about 25°C. The acoustic ECM hydrogel can also be cooled to about 23°C to about 27°C. Acoustic ECM hydrogels can be cooled to approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C to induce the gel phase.

[0065] In some embodiments, acoustic mammalian ECM hydrogels are disclosed, which are thermoreversible and exist in a solid (gel) phase at temperatures below approximately 37°C and in a liquid (sol) phase at temperatures above 37°C. Acoustic hydrogels can be produced using any of the methods disclosed herein. In some embodiments, the storage modulus (G') of the acoustic ECM hydrogel is about an order of magnitude greater than the loss modulus (G”). In further embodiments, the viscosity of the acoustic ECM hydrogel decreases with increasing stress at temperatures from about 15 to about 37°C, e.g., about 15, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and / or 36°C. In further embodiments, the viscosity of the acoustic ECM hydrogel decreases with increasing stress at room temperature, and / or from about 23°C to about 27°C and / or from about 15°C to about 25°C. In one embodiment, the gel-to-sol transition of the acoustic ECM hydrogel occurs at about 37°C, and therefore, since it is sufficiently viscous at body temperature, the hydrogel can be used as a submucosal cushion.

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

[0067] In some embodiments, the acoustic ECM hydrogel contains ECM at concentrations ranging from about 25 mg / ml to about 600 mg / ml. In further embodiments, the acoustic ECM hydrogel contains ECM at concentrations ranging from about 20 mg / ml to about 600 mg / ml, about 25 mg / ml to about 300 mg / ml, about 25 mg / ml to about 200 mg / ml, and about 25 mg / ml to about 150 mg / ml. In further embodiments, the acoustic ECM hydrogel contains ECM in a liquid such as a buffer at concentrations ranging from about 50 mg / ml to about 600 mg / ml. The acoustic ECM hydrogel may also have ECM concentrations ranging from about 50 mg / ml to about 300 mg / ml, about 50 mg / ml to about 200 mg / ml, about 50 mg / ml to about 150 mg / ml, about 50 to 100 mg / ml, or about 100 to 150 mg / ml. In some non-limiting cases, acoustic ECM hydrogels contain ECM at concentrations of approximately 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 mg / ml. In some non-limiting cases, acoustic ECM hydrogels can transport ECM in approximately 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 100-105, 105-110, and 110-115 degrees. It contains ECM at concentrations of 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 mg / ml. Exemplary non-limiting concentrations of ECM also include about 25 mg / ml, 100 mg / ml, and 150 mg / ml. In one non-limiting example, the acoustic ECM hydrogel contains ECM at concentrations ranging from about 25 mg / ml to about 150 mg / ml. In one embodiment, the ECM concentration is about 100 mg / ml.

[0068] In some embodiments, the acoustic ECM hydrogel is at approximately 1400 Pa at 15°C when the ECM concentration is approximately 150 mg / mL. * Viscosity of s, and approximately 400 Pa at a temperature of 25°C. * It has a viscosity of s. In other embodiments, the acoustic ECM hydrogel is approximately 2700 Pa at 15°C when the concentration of ECM is about 150 mg / mL. * At approximately 25°C, the pressure is around 800 Pa. * s and 600 Pa at 37℃ * It has a storage modulus of s.

[0069] The liquid-phase acoustic ECM hydrogel can be placed in a three-dimensional mold before cooling, or spread on a TEFLON® sheet to form a film, see, for example, Figures 1C and 1D. The high concentration of ECM in the acoustic ECM hydrogel (50 to 600 mg / ml) allows for the formation of very thin sheets, e.g., sheets as thin as 4 microns. The acoustic ECM hydrogel can be made in any size greater than 4 microns and in any two-dimensional or three-dimensional shape. In some embodiments, sheets are formed with a thickness of about 4 to about 10 microns, e.g., thicknesses of about 4, 5, 6, 7, 8, 9, or 10 microns. The acoustic ECM hydrogel can be molded 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 open-ended hollow cylinders. Exemplary shapes are shown in Figures 2A–2C. Acoustic ECM hydrogels can also be used as injectable drugs, for example, by placing them in a syringe and extruding them from the syringe in a gel or sol phase.

[0070] In some embodiments, the acoustic ECM hydrogel is absorbed into, adsorbed onto, or dispersed on or within a biocompatible substrate. Non-limiting examples of biocompatible substrates include: meshes, nonwovens, noncellular tissues, polymer compositions, polymer structures, cell proliferation skeletons, implants, orthopedic implants and intraocular lenses, sutures, intravascular implants, stents and transplants. In some embodiments, the substrate is synthetic. In other embodiments, the substrate is natural. The acoustic ECM hydrogel can be imparted or incorporated by any preferred method into nonwoven materials, e.g., bandages, sutures, implants, e.g., implants made of ceramic, metal or polymer, e.g., prostheses, artificial or otherwise modified blood vessels, valves, intraocular lenses or tissue implants. As used herein, the terms “coat” and related terms of the same etymological origin such as “coated” and “coating” refer to a process involving covering an inorganic structure in part or in whole with the compositions described herein. For example, but not limited to, coating inorganic structures with liquid-phase acoustic ECM hydrogels may include methods such as pouring, embedding, layering, dipping, and spraying.

[0071] In another embodiment, a composition containing acoustic ECM hydrogel is coated in liquid phase onto a biocompatible structural material, such as a metal, an inorganic calcium compound such as calcium hydroxide, calcium phosphate, or calcium carbonate, or a ceramic composition. Non-limiting examples of suitable metals include cobalt-chromium alloys, stainless steel alloys, titanium alloys, tantalum alloys, and titanium-tantalum alloys, which may contain both non-metallic and metallic components, such as molybdenum, tantalum, niobium, zirconium, iron, manganese, chromium, cobalt, nickel-aluminum, and lanthanum, and are not limited to various grades of CP Ti (commercially pure titanium) or Ti 6Al 4V (90% wt Ti, 6% wt Al, and 4% wt V), stainless steel 316, Nitinol (nickel-titanium alloy), and titanium alloys coated with hydroxyapatite. Metals are beneficial due to their high strength, flexibility, and biocompatibility. Metals can also be molded into complex shapes, many can withstand corrosion in biological environments, reduce wear, and do not cause tissue damage. Other compositions include ceramics, calcium compounds, and, for example, aragonite, without limitation. Combinations of metals, ceramics, and / or other materials may also be useful.

[0072] Any beneficial agent may be mixed with, co-delivered with, co-administered with, or otherwise combined with any composition described herein. For example, beneficial agents include, but are not limited to, interferons, interleukins, chemokines, monokines, hormones, coagulants, chemotherapeutic agents, and antibiotics.

[0073] How to use Macrophages have been shown to be important regulators in normal healing and tissue development after injury. The disclosed acoustic ECM hydrogels can replicate the effects of the entire ECM on the macrophage phenotype, leading to an increase in M2-like regulatory or remodeling-promoting macrophages. Therefore, any of the compositions disclosed herein can be used to modify the macrophage phenotype, for example, to induce regulatory M2 macrophages.

[0074] In some embodiments, a method is disclosed for inducing M2 macrophages in a subject by administering a therapeutically effective amount of a composition comprising the acoustic ECM hydrogel disclosed herein, thereby inducing M2 macrophages in the subject. In further embodiments, a method is disclosed for reducing M1 (pro-inflammatory) macrophages in a subject. This method comprises administering a therapeutically effective amount of the acoustic ECM hydrogel, thereby inhibiting M1 macrophages in the subject. The subject may be any subject for any purpose, including human and veterinary subjects.

[0075] The disclosed acoustic ECM hydrogel increases hemostasis in injury to the subject. Accordingly, methods for accelerating wound coagulation and / or reducing bleeding time are also disclosed. In some embodiments, hemostasis is induced within about 10 to about 100 seconds after administering the acoustic ECM hydrogel to the subject, for example, within about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 seconds.

[0076] In some embodiments, a therapeutically effective amount of acoustic ECM hydrogel can be administered topically to the site of interest to induce hemostasis. The subject may have a wound. The wound may be an external wound or an internal wound not visible from the outside of the patient. The disclosed acoustic ECM hydrogel is useful as a hemostatic agent for any type of wound. The method may include selecting one of the target subjects, such as a person having any type of wound.

[0077] As shown in Figure 10, acoustic ECM hydrogels shorten the coagulation time. Coagulation can be measured by any method known to those skilled in the art. In the Leewhite test tube method, venous blood is placed in three test tubes and kept at 37°C in a water bath. The coagulation time is determined by tilting the first and then the second test tubes at one-minute intervals, recording the time it takes for a hard clot to form in each of them in sequence, and then detecting the blood clot in the third tube to give the coagulation time. In the capillary method, a glass capillary is filled with blood from a finger puncture. Short pieces of the capillary are cut at regular intervals until a blood clot appears between the cut sections of the capillary. Another method for determining the appearance of a clot is the thromboelastogram method. In this method, a fork is used to move through the blood or plasma sample to sense the viscosity increasing during coagulation. A further method for recording the occurrence of coagulation is by monitoring the translucency of the plasma sample after it has been isolated from the blood. With the appearance of a clot, the sample becomes opaque.

[0078] In some embodiments, methods for treating subjects having inflammation or wounds are disclosed. These methods include topically applying a therapeutically effective amount of acoustic ECM hydrogel to the inflammation or wound. In some non-limiting examples, the subjects have inflammatory disorders, e.g., ulcerative colitis or rheumatoid arthritis, for example, not limited to these. These methods may include applying the ECM hydrogel to a tissue surface. In other non-limiting examples, the subjects are organ transplant recipients, subjects with graft-versus-host disease, subjects with myocardial infarction, or subjects with wounds, e.g., surgical wounds or non-surgical traumatic wounds, for example. Accordingly, methods for accelerating wound healing and / or increasing hemostasis in individuals in need are disclosed, including administering a therapeutically effective amount of a composition comprising the acoustic ECM hydrogel disclosed herein. The administration may be topical, such as at the wound site or on a graft.

[0079] The hydrogel can be applied to any wound site to increase hemostasis and / or increase wound healing. The wound may be a wound of the skin or a wound of any surface, without limitation, including the eye. Methods are also provided for wounds resulting from ischemia and ischemic injury, such as chronic venous leg ulcers caused by impaired and / or insufficient venous return. Thus, the method can utilize topical administration to the dermis or eye. Generally, in these applications, the composition is formulated for topical administration. The hydrogel can be applied to the tissue surface of any organ.

[0080] Topical compositions for the healing of wounds, such as dermal wounds, are disclosed herein. These wounds suitable for treatment may be superficial or deep and may include injuries to the dermis and epidermis of the skin. The wounds may be surgical wounds. Methods are thus provided for promoting wound healing and / or promoting coagulation (increasing hemostasis) in a subject.

[0081] Acoustic hydrogels can be applied directly to a target site, for example, in topical formulations such as sheets and plugs, or as part of a dressing or bandage. Bandages and wound dressings may contain acoustic ECM hydrogels. These can be prepared by applying a gel or liquid-phase acoustic ECM hydrogel to a bandage or wound dressing along with any other desired additives. These sheets, plugs, bandages, or dressings can be used to shorten the coagulation time and / or increase wound healing. Acoustic hydrogels can be administered by injection to a target site, for example, as a solid in a gel phase, or the temperature can be raised above 37°C before administration so that the hydrogel is administered in liquid phase.

[0082] For use in wound treatment and / or to increase hemostasis, acoustic ECM hydrogels typically have concentrations within the ranges described above. Acoustic ECM hydrogels can be applied in a single application. Alternatively, acoustic ECM hydrogels can be applied periodically to the affected area, generally about 1 to 10 times daily, for example, over a period of about 3 to 14 days depending on the nature of the wound. In some cases, it may be desirable to apply the composition indefinitely.

[0083] Acoustic ECM hydrogels affect 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 standard value, the rate of wound healing or hemostasis achieved without treatment, or a control such as treatment with an ECM hydrogel produced by an enzymatic method.

[0084] Acoustic ECM hydrogels can also be used in the treatment of surgical wounds and other intentional interventions, where the composition can be applied immediately after the completion of surgery. Methods are provided for stimulating wound healing and increasing hemostasis at the wound site for surgical wounds, excision wounds, deep wounds including dermal and epidermal injuries, eye tissue wounds, dental tissue wounds, oral wounds, diabetic ulcers, dermal ulcers, elbow ulcers, arterial ulcers, venous congestion ulcers, and burns resulting from heat exposure or chemicals.

[0085] The subjects may be human or mammalian subjects for any purpose, including veterinary subjects. The subjects may be children or adults, e.g., young, middle-aged or older adults. In humans, adult subjects are considered to be over 18 years of age, young adults are generally considered to be between 18 and 35 years of age, middle-aged adults are generally considered to be between 35 and 55 years of age, and older (or aged) human subjects are over 55 years of age, e.g., over 60, 65, 70, 75 or 80 years of age.

[0086] The subjects can heal wounds or injuries at a normal rate. Several diseases and conditions can lead to injury healing. These include diabetes (such as type 2 diabetes mellitus), treatment with both steroids and other pharmacological agents, and ischemic occlusion or injury (such as in peripheral vascular disease or traumatic vascular occlusion). Conditions that induce abnormal wound healing include, but are not limited to, uremia, malnutrition, vitamin deficiencies, obesity, infections, immunosuppression, and complications associated with systemic treatment with steroids, radiotherapy and antineoplastic agents, as well as antimetabolites. Steroids that have been shown to impair wound healing include cortisone, hydrocortisone, dexamethasone, and methylprednisolone. Nonsteroidal compounds such as octreotide acetate have also been shown to impair wound healing (Waddell et al., Am. Surg. 63:446 449, 1997).

[0087] Subjects may have coagulation disorders or be treated with anticoagulants, such as warfarin or PLAAVIX®, without limitation. Subjects may have factor II, factor V, factor VII, factor X, or factor XII deficiencies. Subjects may have hemophilia A, hemophilia B, von Willebrand disease, fibrinogen, or prothrombin deficiencies or structural abnormalities. Therefore, in some embodiments, these subjects are selected for treatment.

[0088] This specification also provides methods for increasing the adhesion of skin grafts to the wound bed and for stimulating re-epithelialization from the wound bed. Types of grafts include, but are not limited to, autologous skin grafts, artificial skin, allogeneic grafts, autologous dermal grafts, autologous epidermal grafts, avascular grafts, Blair-Brown grafts, bone grafts, embryonic tissue grafts, skin grafts, delayed-type grafts, dermal grafts, epidermal grafts, fascial grafts, full-thick grafts, heterologous grafts, xenografts, allogeneic grafts, hyperplastic grafts, laminar grafts, mesh grafts, mucosal grafts, Oria-Tielsch grafts, omental grafts, patch grafts, stalk grafts, penetrating grafts, split skin grafts, and thick split grafts. This method involves administering a therapeutically effective amount of the composition disclosed herein to a subject having a graft, thereby increasing the adhesion and acceptance of the graft and controlling or eliminating bacterial growth. In some embodiments, cells or tissues treated with the composition are transplanted to the subject. In one specific non-limiting example, the composition is administered to the graft, such as a skin graft, prior to transplantation.

[0089] Methods for treating blisters and burns caused by abrasion or chemical injury are also provided. These methods include treatment of skin or internal organs. These methods include, for example, treatment of ovarian injury by treatment with chemotherapeutic agents or cyclophosphamide; cystitis induced by radiation or chemotherapy; or intestinal injury induced by high-dose chemotherapy. The methods include administering a therapeutically effective amount of the compositions disclosed herein to a target to promote the healing of blisters or burns and to reduce or eliminate bacterial growth.

[0090] Methods are provided for promoting the healing of anastomoses and other wounds caused by surgical procedures in individuals. These methods involve administering an effective amount of the composition disclosed herein after and / or during anastomosis or other surgery. Anastomosis is the joining of two tubular structures, for example, when the middle portion of the intestine is removed and the remaining portions are joined to reconstruct the intestinal tract. Unlike skin healing, the healing process of anastomotic wounds is generally indistinct to observe. Furthermore, wound healing occurs rapidly, at least in the gastrointestinal tract, in the absence of complications; however, complications often necessitate correction by additional surgery (Thornton and Barbul, Surg. Clin. North Am. 77:549 573 (1997)). This method promotes the healing of anastomotic wounds. This may include selecting the subjects that are required. The subjects may be subjects whose wound healing is impaired due to one of the above conditions, or subjects that have normal wound healing, for example, subjects that do not have any of the conditions listed above.

[0091] The disclosed acoustic ECM hydrogel is solid at room temperature and transitions to a liquid phase at approximately 37°C. Therefore, in some embodiments, as the hydrogel warms up due to body heat after application to a target, the acoustic ECM hydrogel transitions from a solid phase to a liquid phase over time. In some embodiments, the method may include cleaning the wound to remove the acoustic ECM hydrogel, which can be washed away with a liquid phase.

[0092] In some embodiments, the acoustic ECM hydrogel is sterilized. Sterilization is important to ensure that the acoustic ECM hydrogel of the present invention is sufficiently free from pathogen contamination and suitable for medical use, such as implantation in human or animal bodies. Methods such as gamma irradiation, ethylene oxide, supercritical CO2, hydrogen peroxide gas plasma, or ozone may be suitable for sterilizing the acoustic ECM hydrogel of the present invention, but other sterilization methods known in the art may also be suitable. As shown herein, gamma sterilization is an acceptable sterilization method because the acoustic ECM hydrogel of the present invention maintains its rigidity even after gamma sterilization. In one embodiment, the acoustic ECM hydrogel is gamma sterilized before the ECM solution forms a gel; for example, liquid ECM can be sterilized before sonication or after sonication before gel formation. In another embodiment, the acoustic ECM hydrogel is gamma sterilized after gel formation. In some embodiments, the acoustic ECM hydrogel may remain in gel form or form a gel after sterilization by gamma irradiation.

[0093] In contrast, in the case of enzyme-generated hydrogels, gamma irradiation destabilizes the composition. Therefore, the gel is either not formed or becomes unstable after gamma irradiation.

[0094] Acoustic ECM hydrogel as a submucosal cushion Endoscopy is a procedure that allows examination of the inside of hollow organs or body cavities using an instrument called an endoscope, without the use of invasive surgery. Endoscopy can be used for surgical procedures, such as cauterization of bleeding blood vessels, removal of polyps, adenomas and small tumors, biopsies, or removal of foreign bodies. Endoscopic procedures can be performed in the gastrointestinal tract, respiratory organs, ears, urinary tract, and female reproductive system, as well as inside normally closed body cavities, such as abdominal or pelvic cavities (laparoscopy), joints (arthroscopy), and thoracic organs (thoracoscopy and mediastinoscopy), through small incisions. Endoscopy can be performed in the upper or lower gastrointestinal tract. Endoscopy allows the passage of instruments (such as forceps, electrosurgical knives, endoscopic needles, or scissors) or biooptics to visualize the inside of hollow organs or parts (e.g., bladder, esophagus, stomach, or intestine) for diagnostic or therapeutic purposes. An endoscope is a lit, usually flexible or rigid tubular instrument made of fiber optics, typically having one or more working channels to facilitate sample removal. It includes a suitable lamp and imaging device at its distal end and can be inserted through naturally occurring openings in the body such as the mouth, anus, ears, and nose, or through small surgical incisions. Because a wide variety of body organs or cavities can be examined by endoscopic procedures, there are several specialized endoscopes, such as laryngoscopes, thoracoscopies, capillaries, colonoscopes, sigmoidoscopies, rectoscopes, proctoscopes, anoscopes, arthroscopes, nasaloscopes, laparoscopes, hysteroscopes, neuroscopes, nephroscopes, esophagoscopes, bronchoscopes, gastroscopy, amnioscopy, and cystoscopy.

[0095] Endoscopic procedures are widely applied in the gastrointestinal tract, including the upper and lower gastrointestinal tracts. For example, endoscopic procedures can be used to examine the mucosa lining the gastrointestinal lumen, as well as to detect pathological damage of all sizes, such as inflammatory tissue, polyps, pseudopolyps, serrated lesions, adenomas, ulcers, malformations, preneoplasms and neoplasms, and tumors. Endoscopic procedures can be used for biopsy and removal of pathological damage (polyps, adenomas, malformations, preneoplasms and neoplasms, tumors). Surgical interventions include two types of endoscopic resection procedures commonly used in gastrointestinal endoscopy to remove pathological damage: endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD). These two techniques allow for minimally invasive treatment of gastrointestinal polyps, adenomas, malformations, and early-stage cancers, with a minimal risk of lymph node metastasis.

[0096] This specification discloses a method for incising the mucosa and submucosa from the muscularis propria in a region of an organ of interest. The organ may be the gastrointestinal tract, e.g., the esophagus, duodenum, stomach, small intestine, large intestine (colon), or rectum. The organ may be the bladder, organs of the oral-respiratory system (lungs, throat (pharynx), tongue, nasal passages, sinuses), skin, or uterus and vaginal canal. Examples of specific tissues include respiratory epithelium, nasal epithelium, dermis or epidermal tissue, and uterine epithelium. One exemplary organ is the esophagus. Another exemplary organ is the colon. This method is useful in any organ having mucosa and submucosa in which superficial damage, such as damage from a malignant or precancerous condition, can be formed.

[0097] These methods involve injecting a pharmaceutical composition containing an acoustic ECM hydrogel into the submucosa of the target organ to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the organ. In one embodiment, the organ is not the esophagus. In another embodiment, the organ is the esophagus. The method may be endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD).

[0098] EMR is an endoscopic technique developed for the removal of sessile or flat neoplasms confined to the superficial layers (mucosa and submucosa) of the gastrointestinal (GI) tract. EMR is commonly used for the removal of lesions less than 2 cm in size or for the incisional removal of larger lesions. EMR also plays an important role in the examination of the resected specimen for accurate pathological staging. In contrast to polypectomy, EMR involves lifting the lesion from the muscle layer by injecting a fluid, commonly saline (NS) solution, into the submucosa. EMR is also beneficial for obtaining specimens for accurate histopathological staging to determine the risk of lymph node metastasis. EMR facilitates the complete removal of the affected mucosa by resecting through the central or deeper portions of the submucosa of the intestinal wall. Various EMR techniques have been described, and four methods, including snare resection, are commonly used: (1) injection and cutting method; (2) injection, lift and cutting method; (3) cap-assisted EMR (EMRC); and (4) ligation-assisted EMR (EMRL). In injection and cutting techniques, pathological mucosa is lifted from the muscle layer by forming a submucosal fluid cushion, captured, sphincted using an electrosurgical snare, and then excised. However, injection into the thin submucosa is a delicate process, the injected solution tends to dissipate rapidly, flat and depressed lesions are more difficult to capture with a snare compared to raised lesions, and large or irregularly located lesions may be difficult to remove (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). Injection-assisted EMR is frequently used for large, flat colon polyps. It is frequently used.

[0099] Endoscopic submucosal dissection (ESD) was specifically developed to remove larger lesions. The lesion is incised directly along the submucosa using an electrosurgical knife, even resulting in en bloc resection of large lesions. While ESD is predicted to replace conventional surgery in the treatment of certain cancer stages, it has a higher rate of perforation and bleeding complications than conventional EMR, thus requiring a higher degree of endoscopic skill and experience than EMR. ESD can utilize a variety of electrosurgical knives, such as insulated-tip transmissible knives, needle knives, hook knives, bent knives, triangular-tip knives, flush knives, splash needles, and small-diameter-tip transparent hoods. These knives can be used with high-frequency electrosurgical current (HFEC) generators. ESD is characterized by three steps: (1) injecting a fluid to form a submucosal cushion to lift the injury from the muscle layer; (2) circumferential cutting of the mucosa around the injury; and (3) incision of the connective tissue of the submucosa beneath the injury (see Kakushima et al., Wold J. Gstroenterol. 14(9): 2962-2967, 2008, incorporated herein by reference). Various submucosal injection solutions have been previously developed and shown to satisfy use during EMR, but the introduction of longer ESD procedures required more sustained solutions to help identify the cutting line during submucosal incisions (Uraoka et al., Drug Design, Development and Therapy 2008:2 131-138). The currently disclosed method is: This fulfills the need.

[0100] Submucosal injection is used in EMR because it facilitates the isolation of tissue to be removed immediately before, for example, capture of the target injury with a snare, thereby reducing the risk of thermal damage and perforation and bleeding while also facilitating excision. Submucosal injection plays a crucial role in EMR procedures because the solution must be held in place for a sufficient period and as needed to form a hemispherical shape to facilitate snaring. Furthermore, providing a sufficiently high elevation of the submucosa results in safe submucosal dissection during ESD procedures (Uraoka et al., Drug Design, Development and Therapy). 2008:2 131-138). Furthermore, if inflammation occurs due to the procedure, the treatment site will be retained. The cushion should also possess anti-inflammatory properties. Acoustic ECM hydrogel alleviates stenosis and promotes re-epithelialization. The currently disclosed method also satisfies this need.

[0101] In some embodiments, the disclosed method utilizes an acoustic ECM hydrogel that has anti-inflammatory properties, is inexpensive, non-toxic, easy to inject, and provides a highly persistent submucosal cushion. The acoustic ECM hydrogel is administered to the injection site in its gel state to form a cushion. The cushion can be incised during the procedure so that some of the hydrogel remains on the underlying muscularis propria, thereby aiding healing. The disclosed acoustic ECM hydrogel facilitates the closure of the wound formed by the removal of the excised mucosa / submucosa. In some embodiments, the procedure is ESD. In other embodiments, the procedure is EMR.

[0102] Physiological saline (NS) and its dilution (e.g., ELEVIEW®, U.S. Patent No. 9,226,996, incorporated herein by reference) have been used as submucosal cushions for endoscopic resection. However, the inherent characteristics of these solutions make it difficult to generate a suitable submucosal fluid cushion, maintain the desired height, and retain the cushion in the desired position due to the rapid dispersion of the solution. Furthermore, in ESD, once the mucosa / submucosa is removed, these agents are not retained on the underlying muscularis propria. Moreover, these agents do not aid the healing process, for example, by reducing inflammation. The use of acoustic ECM hydrogels fulfills these needs.

[0103] The acoustic ECM hydrogels disclosed herein can be used in any ESD or ESR. As disclosed in U.S. Patent No. 9,364,580 incorporated herein by reference, an endoscopic injection needle is a device comprising a relatively long catheter, which may be long (up to about 230) cm, in which an internal injection tube with a distal injection needle is slidably positioned. A proximal working handle is connected to the catheter and injection tube to move one relative to the other as needed. Access to the injection tube is generally provided by a leer connector on the handle. The endoscopic injection needle device is generally delivered to the injection site through the working channel of the endoscope. To protect the lumen of the endoscopic working channel from injury, the handle of the injection needle device is operated to pull the distal injection needle into the lumen of the catheter before inserting 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 located at the injection site, its handle is also operated to move the injection needle distally out of the lumen of the catheter. When advanced to its most distal position, the exposed portion of the injection needle is approximately 4-6 mm in length.

[0104] After puncturing the injection site, an acoustic ECM hydrogel, typically contained in a 5ml to 10ml syringe equipped with a Luer lock fitting connected to the needle handle, can be delivered to the injection site, for example, between the submucosa and the underlying muscularis propria, through the injection tube and needle.

[0105] Injection needles and other accessories commonly used during endoscopic procedures, such as snares for polypectomy, clipping instruments, biopsy forceps, and similar devices, are passed through one or more specific channels of the endoscope, usually called the working channel or operating channel. The inner diameter of the working channel can vary considerably depending on the type of endoscope used in GI endoscopy (e.g., gastroscopy, colonoscopy, coloscopy, duodenoscope, sigmoidoscopy, and similar devices). However, the most common endoscopes used in GI endoscopy have working channels with inner diameters ranging from about 2 mm to about 5 mm. Generally, manufacturers of endoscopic accessories produce accessories with outer diameters that allow them to fit all working channels. In some embodiments, endoscopic injection needles have catheter outer diameters ranging from 1.9 mm to 2.3 mm, for example, about 1.9, 2.0, 2.1, 2.2, or 2.3 cm. Thus, considering that the internal injection tube is housed within the external catheter, its inner diameter is typically 1 mm or less. Acoustic ECM hydrogels, disclosed in gel or liquid form, can easily pass through these catheters.

[0106] Acoustic ECM hydrogel can be used in endoscopic resection procedures by aspirating a certain amount of hydrogel from its primary container with a syringe and injecting a suitable amount of the hydrogel with an endoscopic injection needle inserted into the working channel of the endoscope just below the superficial mucosal layer, in order to depose the hydrogel into the submucosa, which will form a cushion once placed in position: the elevation of the mucosal surface allows the endoscopist to perform easy resection of mucosal damage found during the execution of the endoscopic procedure, even if the damage is flat and therefore does not protrude into the lumen, such as the intestinal tract, esophagus, or stomach lumen. At body temperature, acoustic ECM hydrogel is a viscous but fluid gel that transitions to a liquid phase and can be easily injected beneath the superficial mucosal layer to form a cushion for this procedure. Because the gel-sol transition is time-consuming, the cushion remains in place for a sufficient amount of time to perform the resection.

[0107] The presence of at least one dye in the cushion can help the endoscopist visualize the structures beneath the mucosa (e.g., the submucosa and the outer muscular wall), thereby reducing the risk that the endoscopist performing the resection procedure may cause damage to said structures. The use of dye can enable visualization of the cushion cavity and the mucosal base. Removal of damage from the mucosal surface creates a mucosal wound. The persistence of the cushion produced by the injected amount of the pharmaceutical composition allows the endoscopic resection procedure to be performed without the need for reinjection. Acoustic ECM hydrogel is injected submucosa into the target area of ​​the organ of interest, e.g., the area of ​​injury or tumor, to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the organ. The cushion can be incised so that a portion of the acoustic ECM hydrogel is retained in the underlying muscularis propria to aid in the healing process.

[0108] The disclosed method is useful in the esophagus. In an unspecified example, the method includes a method for dissecting esophageal cancer or adenocarcinoma from the esophagus. In another unspecified example, the method includes dissecting the mucosa and submucosa from the esophagus of a subject having Barrett's esophagus. In these embodiments, the acoustic ECM hydrogel may be an acoustic ECM hydrogel of the bladder, small intestinal submucosa (SIS), esophagus, trachea, liver, or dermis.

[0109] The disclosed method may also be useful in other organs. The organ may be any organ of interest, for example, an organ of the gastrointestinal tract. The organ may be in the upper part of the gastrointestinal tract, such as the pharynx, tongue, or mouth. The organ may be the bladder, vaginal canal, or uterus. In some embodiments, the organ may be the colon, duodenum, stomach, cecum, colon, sigmoid colon, rectum, small intestine, or large intestine. In one non-limiting example, the organ may be the stomach, small intestine, or large intestine, and the method includes a method for dissecting a carcinoma or adenocarcinoma from the stomach. In a further non-limiting example, the organ may be the colon, and the method includes dissecting a polyp or carcinoma from the colon. In these embodiments, the acoustic ECM hydrogel may be an acoustic ECM hydrogel of the bladder, submucosa of the small intestine, esophagus, trachea, liver, or dermis.

[0110] As disclosed herein, the acoustic ECM hydrogel is maintained at or below the temperature at which it gels for application as a submucosal cushion.

[0111] The acoustic ECM hydrogel may be maintained at, for example, about 4°C or about room temperature before administration. In one embodiment, the acoustic ECM hydrogel can be administered at a temperature, for example, between 4°C and less than 37°C or between 4°C and 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 utilized. The acoustic ECM hydrogel remains as a gel in the target tissue, which is at a temperature of approximately 37°C. In one embodiment, the gel-to-sol transition of the acoustic ECM hydrogel occurs at approximately 37°C, and therefore the hydrogel is sufficiently viscous at body temperature to be used as a submucosal cushion.

[0112] In some embodiments, the ECM concentration in the hydrogel ranges from 25 mg / ml to about 200 mg / ml, for example, the ECM hydrogel is 25 mg / ml to about 100 mg / ml. In other embodiments, the ECM concentration in the hydrogel is about 50 to about 150 mg / ml, for example, about 75 to about 125 mg / ml, for example, about 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 mg / ml. In a specific non-limiting example, the ECM concentration in the hydrogel is about 100 mg / ml.

[0113] Acoustic ECM hydrogels can be supplied in lyophilized form at room temperature, cold temperature (e.g., about 4°C), or freezing temperature (e.g., about -20°C) and can be restored immediately before administration to the target anatomical region.

[0114] The disclosed method is useful in any subject, including human and veterinary subjects. The subject may be of any age. The subject may be adult or juvenile. In one embodiment, a composition comprising an acoustic ECM hydrogel is injected into target tissue of an organ to form a cushion, which then undergoes an endoscopic surgical procedure, such as an excision, as required. The ECM may be derived from the same species as the subject being treated or from a different species. In some embodiments, the subject is human and the acoustic ECM hydrogel is derived from human or porcine ECM. In other embodiments, the ECM hydrogel is derived from a non-human primate, dog, cat, horse or cattle. The acoustic ECM may be derived from a commercially available donor source. In some embodiments, the acoustic ECM hydrogel may be derived from the tissue of any mammal, e.g., porcine or human tissue, and in some non-limiting examples, the bladder, small intestine or esophagus. Any of the disclosed acoustic ECM hydrogels derived from any donor tissue may be used as a submucosal cushion and / or in any of the disclosed methods. The acoustic ECM hydrogel may be an esophageal acoustic ECM hydrogel or a bladder acoustic ECM hydrogel.

[0115] The disclosed method is invasive because it requires injection to incise the mucosa and submucosa from the muscularis propria of the target intestinal organ region. In some embodiments, the acoustic ECM hydrogel is not applied to the surface of organs such as the gastrointestinal organs, such as the esophagus. The disclosed method can be used in the esophagus, but can also be used in other tissues.

[0116] Any of the methods disclosed herein may include submucosal injection of a pharmaceutical composition containing an acoustic ECM hydrogel into the target organ to form a cushion between the submucosa and the underlying muscularis propria in the area of ​​the organ. Preferred acoustic ECM hydrogels are disclosed above. The acoustic ECM hydrogel gels, incising the mucosa and submucosa from the underlying muscularis propria and inhibiting inflammation in the area of ​​the target organ. The acoustic ECM hydrogel as a gel may be administered endoscopically or by 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 may also be administered endoscopically or by catheter. In further embodiments, the acoustic ECM hydrogel may be an acoustic ECM hydrogel from the bladder, small intestine submucosa, esophagus, trachea, liver, or dermis. In some embodiments, the ECM may be derived from human tissue. In other embodiments, the ECM may be derived from porcine tissue.

[0117] In some embodiments, the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection of the esophagus, and the method includes a method for incising esophageal cancer or adenocarcinoma from the esophagus. In further embodiments, the method includes incising the mucosa and submucosa from the esophagus of a patient with a malformation. In further embodiments, the method includes incising the mucosa and submucosa from the esophagus of a subject with Barrett's esophagus.

[0118] 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 includes a method for incising a polyp, carcinoma, or adenocarcinoma from the colon. In further embodiments, the method includes incising the mucosa and submucosa from the organ of a patient with a malformation. In specific, non-limiting examples, the method includes incising a polyp or carcinoma from the colon.

[0119] The method may also include performing an endoscopic resection procedure on a cushion. In some embodiments, the method includes dividing the cushion so that the hydrogel is retained over the underlying muscularis propria of the esophagus and the mucosa and submucosa are removed from the esophageal region. In some non-limiting embodiments, a portion of the hydrogel cushion retained over the underlying muscularis propria downregulates the activation of pro-inflammatory macrophages in the esophagus.

[0120] This disclosure is illustrated by the following non-limiting embodiments. [Examples]

[0121] Currently, methods for generating hydrogels from ECM include digestion of ECM material with acidic proteases in acidic solutions (Feyetes, Biomaterials 29(11) (2008) 1630-7; Voytik-Harbin, Tissue Engineering 4(2) (1998) 157-174), the use of α-amylase digestion to generate ECM forms (Kommuller et al., JoVE (Journal of Visualized Experiments) (122) (2017) e55436), or the use of chaotropic extraction buffers and laborious dialysis procedures (Uriel, Tissue Eng Part C Methods 15(3) (2009) 309-21; Uriel, Biomaterials 29(27) (2008) 3712-9). ECM hydrogels prepared by such methods inevitably undergo proteolysis and denaturation, which can weaken the overall bioactivity of ECM molecules and tissue-specific ECM components. Furthermore, enzyme-based methods for generating ECM hydrogels require long incubation times of 24–72 hours to achieve sufficient solubilization of ECM components and necessitate 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 enzymatic digestion are also hampered by limited concentration-dependent flow properties (Saldin et al., 2017). To achieve the full clinical capability of ECM hydrogels, a fundamentally different approach has been developed that allows for rapid formation of ECM hydrogels without the use of acidic or alkaline solutions, protease digestion, chemical extraction, or dialysis.

[0122] A method for generating acoustic ECM hydrogels using ultrasonic cavitation, as well as the characterization of the viscoelastic properties, cytocompatibility, and bioactivity of these acoustic ECM hydrogels, is disclosed. In some embodiments, using pulverized ECM as a starting material, the method involves resuspending the ECM in a neutral buffered saline solution and subsequent solubilization using an ultrasonic frequency of 20 kHz. Rapid gelation of the ECM solution can be induced by lowering the temperature of the ECM solution to below 25°C. Gelation time and ECM gel properties can be easily adjusted by adjusting the ECM concentration and the amplitude and duration of sonication. Upon polymerization, the ECM gel is stable at temperatures ranging from 4°C to 37°C. Furthermore, ECM hydrogels prepared using this method are biocompatible and can promote an M2-like remodeling-promoting macrophage phenotype that facilitates downstream constitutive tissue remodeling (Hussey et al., Nature Reviews Materials, 3:159-173 (2018)). These methods offer advantages for the large-scale production of acoustic ECM hydrogels compared to traditional enzymatic methods that produce hydrogels with different properties. Acoustic ECM hydrogels produced by the methods disclosed can be used in tissue engineering and regenerative medicine-based clinical applications.

[0123] (Example 1) material and method 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 prepared by market weight ( Tissue was collected from a pig (Tissue Source Inc.) weighing approximately 110 kg, and the subcutaneous fat and epidermis were removed by mechanical exfoliation. This tissue was then treated with 0.25% trypsin (Thermo Fisher Scientific) for 6 hours, 70% ethanol for 10 hours, 3% H2O2 for 15 minutes, 1% Triton X-100 (Sigma-Aldrich) in 0.26% EDTA / 0.69% Tris for 6 hours, with solution changes for a further 16 hours, and 0.1% peracetic acid / 4% ethanol (Rochester Midland) for 2 hours. After the final step, alternating water washes with water and phosphate-buffered saline (PBS) washes were performed between each chemical exchange. All chemical exposures were carried out under agitation on an orbital shaker at 300 rpm. The dermal ECM was then lyophilized and pulverized to fine particles using a Wiley mill on a #60 mesh screen.

[0124] Preparation of bladder matrix (UBM): The UBM was prepared as previously described (Mase VJ, et al. Orthopedics. 2010; 33(7):511). Pig bladders from market weight animals were obtained from Tissue Source, LLC. Briefly, the serosal layer, muscularis exomucosa, submucosa, and muscularis mucosa were mechanically removed. The luminal urothelial cells of the mucosa were separated from the basement membrane by washing with deionized water. The remaining tissue consisted of the basement membrane and the underlying lamina propria of the mucosa, which was decellularized by stirring at 300 rpm for 2 hours in 4% ethanol and 0.1% peracetic acid. The tissue was then broadly washed with PBS and sterile water. The UBM was then lyophilized and finely ground to fine particles using a Wiley mill on a #60 mesh screen.

[0125] Preparation of the small intestinal submucosa (SIS): The SIS was prepared as previously described (Badylak SF, et al. J Surg Res. 1989; 47(1):74-80). Briefly, the jejunum was taken from 6-month-old market-weight pigs (approximately 110 to 120 kg) and split longitudinally. The superficial layer of the mucosa was mechanically removed. Similarly, the serosal and muscular layers were mechanically removed, leaving the submucosa and basal portions of the mucosa. Decellularization and disinfection of the tissue were completed by stirring at 300 rpm for 2 hours in 4% ethanol and 0.1% peracetic acid. The tissue was then broadly washed with PBS and sterile water. The SIS was then lyophilized and finely ground to a fine particle using a Wiley mill on a #60 mesh screen.

[0126] Preparation of esophageal ECM: The esophageal ECM was prepared as previously described (Keane TJ, et al. al. Tissue Eng Part A. 2015;21(17-18):2293-300). In short, esophageal ECM ( eECM) mechanically separates the mucosa and submucosa from the muscular layer, and the mucosal layer is treated on a rocker plate with 1% trypsin / 0.05% EDTA (Invitrogen, Carlsbad, CA) at 37°C for 1 hour, followed by 15 minutes in deionized water and 1M sucrose (Fisher). The esophageal ECM was prepared by exposing it to 100 U / mL DNase (Invitrogen) on a rocker plate for 2 hours, followed by washing with PBS, deionized water, PBS, and deionized water for 15 minutes. All washing solutions were stirred at 300 rpm on a shaker plate. The esophageal ECM was then lyophilized and finely ground using a Wiley mill on a #60 mesh screen.

[0127] ECM Sonication: 100 mg of ECM powder was resuspended in phosphate-buffered saline (PBS) in a 15 mL conical tube and sonicated using a FISHERBRAND® Model 120 Sonic Dismembrator equipped with a 1 / 8” probe. The ECM concentration was varied from 20 to 200 mg (w / v). The resuspended ECM was sonicated with pulses of 30 seconds on and 45 seconds off, set to 100% amplitude. The cycle was repeated six times to produce a soluble ECM solution. This periodic pulse setting ensured that the gel solution maintained a temperature of 34–40°C.

[0128] Gelation of sonicated ECM solution: The ECM solution was poured into a 3D mold, and the temperature was lowered to 25°C or below to induce gelation. The ECM gel was stored at 4°C, or freeze-dried to produce a lyophilized ECM construct that maintained its 3D geometric structure. Alternatively, the ECM solution was spread uniformly 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 the water, resulting in an ultrathin ECM sheet.

[0129] Preparation of ECM putty: ECM powder was resuspended in 25 mg / ml (w / v) PBS and sonicated as described above. At a concentration of ≤25 mg / ml and a temperature between 4 and 30°C, the solubilized ECM forms a putty.

[0130] ECM Hydrogel Fluid Dynamics: All fluid dynamic data are as previously described (Medberry CJ, et al. Biomaterials. 2013; 34(4):1033-40) 40mm parallel plate geometry Samples were collected using a structured rheometer (AR2000, TA instruments, New Castle, DE) and analyzed using the American Society for Testing and Materials (ASTM) standard F2900-11 (Guide for Characterizing Hydrogels Used in Regenerative Medicine). Temperature was controlled within 0.1°C using a Peltier plate. Gel precursors were loaded onto a parallel plate rheometer at room temperature (25°C) or 4°C. Mineral oil was used to seal the edges of the sample-plate interface and minimize sample evaporation.

[0131] Scanning electron microscopy: Scanning electron microscopy images were taken to examine the surface topology of the ECM hydrogel. Samples were fixed in cold 2.5% (v / v) glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA) for at least 24 hours, followed by three washes in PBS. The fixed samples were then dehydrated using a stepwise series of alcohols (30, 50, 70, 90, 100%) for 15 minutes each, followed by 15 minutes in hexamethylenediamine (Fisher), 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 at 100× and 500× magnification using a JEOL JSM6330f scanning electron microscope (JEOL, Peabody, MA).

[0132] Cytocompatibility Assay: 3T3 fibroblasts were seeded on 96-well plates coated with ECM hydrogel prepared from UBM, SIS, or dermis. Uncoated wells were used as controls. Cells were cultured in Dulbecco's Modified Minimal Essential Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 24 hours after cell seeding, the VYBRANT® MTT cell proliferation assay kit (Thermo Fisher) was used to assess cell viability according to the manufacturer's protocol. The absorbance of the converted dye was measured at a wavelength of 540 nm.

[0133] Hemostasis assay. The Liewhite coagulation assay was used. Fresh whole blood was collected in a test tube, and the tube was repeatedly tilted until coagulation was observed.

[0134] Liver laceration model: 6-8 week old SD rats were anesthetized with isoflurane (1-3%). The animals were maintained at surgical level anesthesia with 1.5-2.5% isoflurane in oxygen and placed in a lateral abdominal position. A small incision was made using sterile equipment, and a 2 cm midline laparotomy was performed below it to expose the liver. A 2 mm deep and 5 mm long incision was made on the ventral surface of the liver by placing the blade of a #11 scalpel on Kelly forceps so that 2 mm of the blade was exposed. The wound was allowed to bleed for 3 seconds, and then wiped clean with sterile gauze. The test specimen was then placed on the defect site, and the coagulation time was recorded.

[0135] Macrophage activation: Mouse bone marrow was collected from 6-8 week old B6 mice. Cells collected from the bone marrow were washed and cultured on plates at 2 × 10⁶ cells / mL. They were differentiated into macrophages for 7 days in the presence of macrophage colony-stimulating factor (MCSF), with the medium completely changed every 48 hours. Macrophages were then activated for 24 hours with one of the following: 1) 20 ng / mL interferon-γ (IFNγ) and 100 ng / mL lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) to promote the MIFNγ+LPS phenotype (M1-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 an incubation period at 37°C, the cells were washed with sterile PBS and fixed with 2% paraformaldehyde (PFA) for immunolabeling. To prevent nonspecific binding, the cells were incubated at room temperature for 1 hour in a blocking solution consisting of PBS, 0.1% Triton-X, 0.1% Tween®-20, 4% goat serum, and 2% bovine serum albumin. The blocking buffer was then removed, and the cells were incubated with the primary antibody. The cells were incubated at 4°C for 16 hours, the primary antibody was removed, and the cells were washed with PBS. A solution of fluorescent-conjugated secondary antibody was added to the wells at room temperature for 1 hour. The antibody was then removed, the cells were washed with PBS, and the nuclei were counterstained using DAPI. Cytokine-activated macrophages were used to establish a standardized exposure time (positive control), which was then kept constant across the group.

[0136] (Example 2) result A method has been developed for preparing hydrogels from extracellular matrix (ECM). Using non-cellular tissue as the starting material, the sonication technique can be applied to many tissue-specific ECMs, including the dermis, bladder matrix (UBM), and small intestinal submucosa (SIS). This approach involves resuspending the pulverized ECM in a neutral buffered saline solution and then solubilizing the ECM using an ultrasonic frequency of 20 kHz with amplitudes ranging from, for example, 20–100% (Figure 1). After 60 seconds of sonication, rapid gelation of the ECM solution is induced by lowering the temperature of the ECM solution to below 37°C (Figures 4, 5, and 6). Results from hydrodynamic evaluations show that ECM hydrogels can be prepared using this method with ECM concentrations ranging from 25 mg / ml to 150 mg / ml (Figures 7 and 8). Upon polymerization, the ECM gel is stable at room temperature and can adapt to a customizable 3D geometric structure (Figure 2). ECM hydrogels prepared by sonication were shown to be cytocompatible when used as a substrate for in vitro cultured cells (Figures 9 and 13). Scanning electron micrographs of the gels show a high-density fibrous network (Figure 3). Furthermore, ECM hydrogels can be used as hemostatic agents that can be applied to anatomical sites in patients to aid in hemostasis (Figures 10 and 11). The results are provided in the attached figures.

[0137] (Example 3) Materials and methods for Examples 4-7 Preparation of ECM biological skeleton: Porcine dermal 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. This tissue was then treated with 0.25% trypsin (Thermo Fisher Scientific) for 6 hours, 70% ethanol for 10 hours, 3% H2O2 for 15 minutes, 1% Triton X-100 (Sigma-Aldrich) in 0.26% EDTA / 0.69% Tris for 6 hours, with solution changes for a further 16 hours, and 0.1% peracetic acid / 4% ethanol (Rochester Midland) for 2 hours. After the final step, alternating water washes with water and phosphate-buffered saline (PBS) washes were performed between each chemical exchange. All chemical exposures were carried out under agitation on an orbital shaker at 300 rpm. The dermal ECM was then freeze-dried and finely ground to fine particles using a Wiley mill with a #40 mesh screen.

[0138] Porcine bladder matrix (UBM) was prepared as previously described (Mase et al., Orthopedics 33(7):511 (2010)). Briefly, the serosal layer, muscularis exomucosa, submucosa, and muscularis mucosa were mechanically removed. The luminal urothelial cells of the mucosa were separated from the basement membrane by washing with deionized water. The remaining tissue consisted of the basement membrane and the underlying lamina propria of the mucosa, which was decellularized by stirring at 300 rpm for 2 hours in 4% ethanol and 0.1% peracetic acid. The tissue was then broadly washed with PBS and sterile water. The UBM was then lyophilized and finely ground using a Wiley mill on a #40 mesh screen.

[0139] The submucosa (SIS) of the small intestine of pigs was prepared as previously described (Badylak et al., J Surg Res 47(1) (1989) 74-80). In short, the jejunum was prepared from market weight of pigs at 6 months of age. The tissue was collected from pigs weighing approximately 110 to 120 kg and split lengthwise. The superficial layer of the mucosa was mechanically removed. Similarly, the serosal layer and muscular layer were mechanically removed, leaving the submucosa and basal portion of the mucosa. Decellularization and disinfection of the tissue were completed by stirring at 300 rpm for 2 hours in 4% ethanol and 0.1% peracetic acid. The tissue was then broadly washed with PBS and sterile water. The SIS was then freeze-dried and finely ground to fine particles using a Wiley mill with a #40 mesh screen.

[0140] Porcine esophageal ECM was prepared as previously described (Keane et al., Tissue Eng Part A 21(17-18) (2015) 2293-300). In short, esophageal extracellular membrane filtration (eECM) involves mechanically separating the mucosa and submucosa from the muscular layer, and then rinsing the mucosa on a rocker plate in 1% trypsin / 0.05% EDTA (Invitrogen, Carlsbad, CA) at 37°C for 1 hour, in deionized water for 15 minutes, in 1M sucrose (Fisher Scientific, Pittsburgh, PA) for 30 minutes, in deionized water for 30 minutes, in 3.0% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 48 hours, in deionized water for 15 minutes, in phosphate-buffered saline (PBS; Fisher Scientific) for 15 minutes, in 10% deoxycholic acid (Sigma-Aldrich) for 4 hours, in deionized water for 30 minutes, and in 0.1% peracetic acid in 4.0% ethanol (Rochester Midland Corp., Rochester, NY) for 4 hours, at a concentration of 100 U / mL on a rocker plate. The eECM was prepared by exposing it to DNase (Invitrogen) for 2 hours, followed by washing with PBS, deionized water, PBS, and deionized water for 15 minutes. All washing solutions were stirred at 300 rpm on a shaker plate. The eECM was then freeze-dried and finely ground to fine particles using a Wiley mill on a #40 mesh screen.

[0141] Porcine tracheal extracellular membranes (TCMs) were prepared as previously described with minor modifications (Lange et al., Journal of tissue engineering and regenerative medicine 11(3) (2017) 800-811). Briefly, tracheas were incubated for 30 minutes in a washing solution containing 0.25% Triton X-100 + 0.25% sodium deoxycholate under negative pressure vacuum cycles (15 cycles, -0.95 kPa maximum vacuum), followed by overnight immersion in fresh washing solution. This process was repeated daily, with the washing solution replaced with sterile DI water on days 2 and 3, with a solution of 2000 KU / ml DNase in water on day 4, and with sterile DI water on day 5. This cycle was repeated once during a total of 10 days of vacuum cycles, followed by overnight sterilization in 15% peracetic acid + 4% ethanol, and then washing and storage in sterile PBS. The tracheal ECM was then freeze-dried and finely ground into particles using a Wiley mill with a #40 mesh screen.

[0142] Porcine liver extracellular matrix (LECM) was prepared as previously described (Loneker et al., Journal of Biomedical Materials Research Part A 104(4) (2016) 957-965). Liver was collected from market-weight pigs (110-130 kg). Tissue was extracted using a scalpel. 5cm 3 The liver tissue was cut into sections and washed three times in deionized water for 15 minutes each by mechanical agitation on an orbital shaker. The sections were then gently kneaded to aid cell lysis and immersed in 0.02% trypsin / 0.05% EGTA at 37°C for 2 hours. The tissue was washed with Type 1 water, kneaded repeatedly, and then mechanical agitation of the liver sections continued in 3% Triton X-100 for 18-24 hours. Washing was repeated until all visible residue of cellular material was removed. After treatment, the liver ECM was immersed in a 0.1% peracetic acid solution and then washed repeatedly with Type 1 water or PBS at pH 7.4. The liver ECM was then freeze-dried and finely ground into particles using a Wiley mill on a #40 mesh screen.

[0143] Solubilization of ECM by ultrasonic cavitation: The pulverized ECM was resuspended in 10 ml of 1 × phosphate-buffered saline (PBS) in a 50 mL conical tube and sonicated using a FISHERBRAND® Model 120 Sonic Dismembrator equipped with a 1 / 8” probe. The ECM concentration was varied from 25 to 100 mg / ml (w / v), and the sonication time was varied from 30 to 500 seconds at amplitudes ranging from 20% to 100%. An example of the experimental setup is shown in Figure 14.

[0144] Collagen and sGAG quantification: Grinding dECM (100 mg / ml) was sonicated, and the sample was centrifuged at 10,000 × g for 30 minutes to compress the insoluble ECM components. The clear supernatant containing the solubilized ECM components was transferred to a new tube. The collagen concentration of the supernatant solution was determined using the Sircol assay kit (Biocolor Ltd., UK) according to the manufacturer's recommended protocol. The sulfated glycosaminoglycan (sGAG) concentration was determined using the Blyscan sulfated glycosaminoglycan assay kit (Biocolor Ltd., UK) according to the manufacturer's recommended protocol.

[0145] Gelation assay: To measure gelation time, use the test tube inversion method (Quin et al., Frontiers in chemistry 6 (2018); El-Fiqi et al., Acta biomaterialia 9(12)). (2013) 9508-9521) was used. Sample material (25, 50 or 100 mg / ml) Immediately after sonication, 0.5 ml of the sample was transferred to a test tube and incubated at a constant temperature of 4°C or 25°C. The fluidity of the sample was observed every minute by inverting the tube. The time at which the flow of the sample stopped was taken as the gelation time and recorded.

[0146] Scanning electron microscopy: Scanning electron microscopy images were taken at 50 and 100 mg / ml to examine the surface topology of the dECM hydrogel. Samples were fixed in cold 2.5% (v / v) glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA) for at least 24 hours, followed by three washes in PBS. The fixed samples were then dehydrated using a stepwise 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 using a JEOL JSM6330f scanning electron microscope (JEOL, Peabody, MA).

[0147] Viscoelasticity measurements: All hydrodynamic data were obtained as previously described (Medberry, et al., Biomaterials 34(4) (2013) 1033-40) using 40mm parallel plates with geometric structure attached. Samples were collected using an rheometer (AR2000ex, TA Instruments, New Castle, DE) and analyzed using the American Society for Testing and Materials (ASTM) standard F2900-11 (Guide for Characterizing Hydrogels Used in Regenerative Medicine). For each temperature profile tested, samples of each tissue type (dECM and eECM, 100 mg / ml) were brought to the starting temperature (4, 25, or 37°C) for one hour prior to the test. Samples were loaded onto an AR-2000ex rheometer with a 40 mm parallel plate geometry set to the starting temperature. Mineral oil was used to seal the edges of the sample-plate interface to minimize evaporation during the test. To measure the gelation kinetics of the sonicated hydrogels, a vibration time sweep was performed for one hour by applying a small 0.5% vibration tension at a frequency of 1 rad / second and rapidly changing the temperature according to the temperature profile tested (37 or 4°C). Data were exported using Trios software (TA Instruments) and analyzed using Prism v8 software (GraphPad, San Diego, CA). "Average storage modulus" was the average storage modulus G' over the last 10 minutes of a 60-minute test, representing the G' plateau. Time to 50% gelation was determined as the time to 50% of the average storage modulus.

[0148] In vitro metabolic assay: 3T3 fibroblasts were seeded on 96-well plates coated with 100 mg / ml ECM hydrogel prepared from UBM, SIS, or dECM. Uncoated wells were used as controls. Cells were cultured in Dulbecco's Modified Minimal Essential Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 24 hours after cell seeding, the Vybrant® MTT cell proliferation assay kit (Thermo Fisher) was used to assess cell viability according to the manufacturer's protocol. The absorbance of the converted dye was measured at a wavelength of 540 nm.

[0149] 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 onto 6-well plates coated with 100 mg / ml ECM hydrogel prepared from UBM or dECM. Uncoated wells were used as controls. 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. Images were taken for 5 × 200× fields over 3 technical replicates. CellProfiler was used to quantify the percentage of live and dead cells. Images were taken with a Zeiss Axiovert microscope that captures 5 random fields at 200× magnification. Quantification of the percentage of live and dead cells was completed using a custom CellProfiler pipeline.

[0150] In vitro macrophage response: Mouse bone marrow cells were collected from 6- to 8-week-old B6 mice. Cells collected from the bone marrow were washed and plated at 2 × 10 6 cells / mL and differentiated into macrophages for 7 days in the presence of macrophage colony-stimulating factor (MCSF), with the medium being completely replaced every 48 hours. Next, macrophages were activated for 24 hours with one of the following: 1) 20 ng / mL interferon-γ (IFNγ) and 100 ng / mL lipopolysaccharide (LPS) (Affymetrix eBioscience, Santa Clara, CA; Sigma Aldrich) that promote the IFNγ+LPS M1-like phenotype; 2) IL-420 ng / mL interleukin (IL)-4 (Invitrogen) was used to promote the phenotype (M2-like); 3) 2 mg / mL dECM hydrogel, or 4) 2 mg / mL eECM hydrogel. After a 24-hour incubation period at 37°C, cells were washed with sterile PBS and fixed with 2% paraformaldehyde (PFA) for immunolabeling. To prevent nonspecific binding, cells were incubated at room temperature for 1 hour in a blocking solution consisting of PBS, 0.1% Triton-X, 0.1% Tween-20, 4% goat serum, and 2% bovine serum albumin. The blocking buffer was then removed, and cells were incubated with the primary antibody. Cells were incubated at 4°C for 16 hours, the primary antibody was removed, and cells were washed with PBS. A solution of fluorescent-conjugated secondary antibody was added to the wells at room temperature for 1 hour. The antibody was then removed, cells were washed with PBS, and nuclei were counterstained using DAPI. Cytokine-activated macrophages (positive control) were used to establish a standardized exposure time (positive control) for imaging the remaining treatment groups.

[0151] Statistical Methods: All analyses were performed using Prism software (GraphPad Software Inc.) with significance defined as p<0.05. Solubilized collagen and sGAG results were analyzed using ANOVA and pairwise comparisons with post-hoc Tukey's multiple comparison test. Gelation time results were analyzed using ANOVA with post-hoc Tukey's multiple comparison test. Student's unpaired t-tests were performed for single comparisons. Hydrodynamic data were analyzed by two-way ANOVA for independent variables temperature and ECM type and dependent variable storage modulus, and post-hoc Tukey's multiple comparison test for the main effect of temperature. Student's unpaired t-tests were performed to compare gelation times for dECM and eECM.

[0152] (Example 4) Solubilization of collagen and sGAG To evaluate the effect of sonication amplitude on the solubilization of collagen and sulfated glycosaminoglycans (sGAGs), pulverized dECM was sonicated for 300 seconds at 20%, 40%, 60%, 80%, and 100% amplitude. The results showed a significant increase in solubilized collagen with increasing sonication amplitude (Figure 15A). In contrast, sonication amplitude did not have a significant effect on sGAG solubilization (Figure 15B). To evaluate the effect of sonication time on collagen sGAG solubilization, pulverized dECM was sonicated at 100% amplitude for times ranging from 30 to 500 seconds. The results showed a significant increase in solubilized collagen with increasing sonication time (Figure 15C). In contrast, sonication time did not have a significant effect on sGAG solubilization (Figure 15D).

[0153] (Example 5) Gelation kinetics and qualitative research The effects of sonication time and amplitude on the gelation kinetics of dECM hydrogels prepared at concentrations of 25, 50, or 100 mg / ml were calculated. The results showed that for all concentrations tested, incubation of the pre-gel solution at 4°C compared to 25°C significantly reduced the time required for gel formation (Figure 16A). Furthermore, when incubated at 4°C, the 100 mg / ml concentration showed a significant reduction in gelation time compared to the 25 mg / ml concentration (Figure 16A). For all concentrations tested, increasing the sonication amplitude from 20 or 40% to 80 or 100% resulted in a significant reduction in gelation time (Figure 16B). A significant difference in gelation time was observed between 25 and 100 mg / ml concentrations sonicated at 40 or 100% amplitude (Figure 16B). Gelation kinetics were determined for UBM, SIS, eECM, tECM, and LECM prepared at a concentration of 100 mg / ml (Figure 16C). The results showed that for all ECM tissue types tested, incubation of the pre-gel solution at 4°C compared to incubation at 25°C significantly reduced the time required to form the gel. Furthermore, eECM hydrogels showed a significantly reduced gelation time at 4°C compared to all other ECM tissue types. When incubated at 25°C, eECM showed a significantly reduced gelation time compared to tECM and LECM. Scanning electron micrographs of 50 mg / ml and 100 mg / ml dECM hydrogels showed a high-density fibril network due to organized collagen fibrils (Figure 3).

[0154] (Example 6) Hydrodynamic measurements Two tissue types of ECM hydrogels (dECM and eECM, 100 mg / ml) were tested under the following three temperature profiles: 1) 4→37°C, 2) 25→37°C, and 3) 37→4°C (Figure 17A). When the temperature was decreased (37→4°C), the ECM hydrogels showed an S-shaped increase in gelation (storage modulus, G'). At the plateau of the S-shaped gelation curve, the hydrogels showed storage modulus G' > loss modulus G''. When the temperature was increased (4→37°C or 25→37°C), the stiffness of the hydrogels was maintained over time (G'>>G'') (Figure 17A). When the final temperature was increased, the storage modulus G' increased in dECM hydrogel when the final temperature was rapidly decreased from 37 to 4°C (3447.3±3340.1Pa) compared to when the final temperature was increased from 4 to 37°C (234.4±215.7Pa) (p=0.04) or from 25 to 37°C (245.8±94.5Pa) (p=0.04) (Figure 17B). When the final temperature was rapidly decreased from 37 to 4°C (2237.4±227.1Pa), the stiffness of eECM also showed an increasing trend compared to when the final temperature was rapidly decreased from 4 to 37°C (733.0±363.7Pa) or from 25 to 37°C (624.1±133.5Pa), but this was not statistically significant (p=0.4) (Figure 17B). For dECM and eECM, the gelation time (time to 50% gelation) was determined for the S-shaped gelation profile from 37°C to 4°C (Figure 18C). Student's unpaired t-test showed that the gelation time was shorter for eECM (0.5±0.4 mins) compared to dECM (2.5±0.5 mins) (p=0.006). Since gelation was maintained, the gelation time was not determined for the temperature profile when the temperature was raised to 37°C.

[0155] (Example 7) in vitro cellular response The MTT cell proliferation assay demonstrated that ECM hydrogels prepared from dECM, UBM, or SIS were not cytotoxic to NIH 3T3 fibroblasts (Figure 18A). Similarly, results from viability / death assays showed that primary equine mesenchymal stem cells retained nearly 100% viability when seeded on ECM hydrogels prepared from dECM or UBM (Figures 18B, C). There were no differences in proliferation and viability between these treatments and when compared to cells cultured for 24 hours on tissue culture plastic (control) (Figures 18B, C). ECM hydrogels prepared using pepsin digestion methods have been previously shown to promote the 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). EC prepared using ultrasonic cavitation method. To evaluate whether M hydrogels exhibit similar effects on macrophages, primary mouse bone marrow-derived macrophages were stimulated with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) to induce an M1-like macrophage phenotype, and with interleukin-4 (IL-4) to induce an M2-like phenotype, dECM hydrogel, or eECM hydrogel. All experimental groups showed homogeneous F4 / 80 staining. Controls showed the expected increase in iNOS when macrophages were treated with IFNγ / LPS, and an increase in Fizz1 when treated with IL-4 (Figure 18B, D, E). As indicated by the concomitant Fizz1 expression with minimal iNOS expression, dECM and eECM hydrogel treatment was found to promote M2-like macrophage activation similar to that of IL-4-treated macrophages (Figure 18B, D, E).

[0156] Therefore, we evaluated the gelation kinetics, hydrodynamic properties, cytocompatibility, and bioactivity of ECM hydrogels prepared using ultrasonic cavitation. While this study primarily focused on the use of dECM to develop and evaluate ultrasonic cavitation methods, we also used ECM from five additional donor tissues in selective assays to demonstrate that ultrasonic cavitation methods can be applied to ECM derived from a wide range of non-cellularized tissues, as summarized in Table 1. [Table 1]

[0157] In this study, the ECM skeleton was solubilized without the need for digestion by acidic proteases in acidic solutions or the use of chaotropic extraction buffers and dialysis procedures, which can adversely affect the molecular composition of the ECM. When incubated at temperatures of 25°C or below, the sonicated ECM self-assembled into a gel. While not constrained by theory, gelation may be due to the presence of self-assembling molecules such as collagen. Indeed, sonication of the ECM skeleton material resulted in a considerable increase in solubilized collagen with increasing sonication time and amplitude. Unlike ECM hydrogels prepared using pepsin digestion, which remained liquid at 25°C and gel at 37°C, hydrogels prepared using the ultrasonic cavitation method formed a stable gel when the temperature was lowered to 25°C or below. The thermomechanical properties of sonicated ECM are similar to those reported for hydrolyzed collagen, which can form a gel after cooling to temperatures below 30°C (Tosh et al., Applied Physics Letters 84(21) (2004) 4242-4244). However, recent studies using circular dichroism analysis, atomic force microscopy, and FTIR on collagen extracted from bovine tendons have shown that the triple helix structure of collagen remains intact and unaffected by sonication (Li et al., Sonochemistry 16(5)(2009) 605-609). Similarly, the same applies to Lateolabrax japonicus. Extraction of collagen from skin showed that 3 hours of sonication at 80% amplitude did not induce any detectable changes in the structural integrity of collagen molecules (Kim et al., Fisheries Science 79(5) (2013) 849-856). In this study, scanning electron microscopy images of sonicated ECM showed a high-density fibrous network of organized collagen fibrils. Furthermore, despite the inverse relationship between gelation and temperature of ECM hydrogels produced using ultrasonic cavitation, the stiffness of the hydrogel was maintained over time when the temperature was increased (4 → 37°C or 25 → 37°C) (G'>>G). These findings suggest that the gelation process of sonicated ECM is not simply a product of a collagen chemical reaction, but rather a result of interactions between various components within the solubilized ECM, including laminins and other self-assembling molecules such as proteoglycans.

[0158] NIH 3T3 fibroblasts and primary equine mesenchymal stem cells were able to adhere to and proliferate on ECM hydrogels generated using sonic cavitation. Furthermore, although the mechanism(s) of ECM-mediated tissue remodeling are only partially understood, the activation state of invasive macrophages from a pro-inflammatory M1-like phenotype to a constructive and remodeling-inducing M2-like macrophage phenotype at remodeling sites was shown to be a predictor of favorable downstream remodeling outcomes (Brown et al., Acta Biomater). 8(3) (2012) 978-87). The results presented herein demonstrate that ECM hydrogels generated using ultrasonic cavitation retain their ability to promote the M2-like macrophage phenotype.

[0159] (Example 8) Gamma-ray irradiation of acoustic hydrogels Acoustic hydrogels (100 mg / mL) were sterilized by 20 kGy gamma irradiation at room temperature. Hydrogel "stiffness" over time was measured for gamma-irradiated (20 kGy) and non-sterilized control acoustic hydrogels (dermal ECM 100 mg / mL). Storage modulus ("stiffness") (G') and loss modulus (G") were measured by applying a small 0.5% vibrational tension to the sample. Three temperature profiles were tested: temperature was rapidly increased from the initial storage temperature to the final temperature: 4 to 37°C, 25 to 37°C, or 37 to 4°C. (Figure 20A) A representative graph of the time sweep is shown. (Figure 20B) The average storage and loss moduli averaged over the final 5 minutes of the test are shown.

[0160] After the formation of the acoustic hydrogel, the gel was placed in a cesium-137 irradiator and exposed to ionizing radiation at 2065 rads / min at room temperature for 16 hours, resulting in a final radiation dose of 20 kGy.

[0161] Surprisingly, and unexpectedly, gamma irradiation did not affect the acoustic hydrogel's ability to maintain its gel shape. In contrast, enzymatically produced ECM hydrogels could not remain as gels when gamma-irradiated, and gamma-irradiated pre-gels of ECM hydrogels could not form a gel when gamma-irradiated before gelation.

[0162] (Example 9) Acoustic hydrogels as submucosal fluid cushions Acoustic hydrogels evaluated for use as submucosal cushions 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) in a 50 mL conical tube. The samples were sonicated at 100% amplitude for 3 minutes using a FISHERBRAND® Model 120 Sonic Dismembrator equipped with a 1 / 8” probe. After sonication, the samples were transferred to a 5 ml syringe and incubated at 4°C to induce gel formation.

[0163] Anesthesia was induced with acepromazine (0.01 mg / kg, SC) and ketamine (5-11 mg / kg), and surgical-level anesthesia was maintained with 1-5% isofluorane administered via endotracheal tube. Throughout the procedure and the immediate postoperative period, pigs were administered 2 ml / kg / h of lactated Ringer's solution IV. Temperature was controlled via a warm water recirculation heating pad placed beneath the animals. Physiological parameters such as heart rate, respiratory rate, body temperature, and responsiveness were monitored during the procedure. Antibiotic prophylaxis with 25 mg / kg cefazolin was administered before commencing the procedure.

[0164] To evaluate the mucosa of tubular organs, pigs were placed in a supine position and a Pentax EG3430K endoscope was used. After identifying reference points within the organs, the mucosa and submucosa were separated from the underlying layers at the resection site by injecting 8 mg / ml of blue-stained gel-type acoustic ECM hydrogel into the submucosal space using an Olympus Injectorforce 4mm 23G needle to provide visual contrast. Approximately 2-5 ml of blue gel was injected per site. The entire circumference (100%) of the mucosa was removed for a length of 5 cm using band-ligation EMR technique. A Cook Duette kit with ligation bands was used for EMR. The mucosa was then resected using a snare. The results are shown in Figures 19A-19C.

[0165] Given the many possible embodiments to which the principles of our invention can be applied, it should be recognized that the exemplary embodiments are merely examples of the invention and should not be considered to limit the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, we claim all within the scope and spirit of these claims as the invention. In certain embodiments, for example, the following items are provided: (Item 1) A method for producing an extracellular matrix (ECM) hydrogel, comprising solubilizing mammalian ECM in a liquid using an ultrasonic frequency to produce a liquid-phase acoustic ECM hydrogel. (Item 2) The method according to item 1, wherein the ultrasonic frequency is applied at a temperature between 30°C and 43°C. (Item 3) The method according to item 1 or 2, wherein the ultrasonic frequency is applied at a temperature between 35°C and 40°C. (Item 4) The method according to any one of items 1 to 3, wherein the ultrasonic frequency is applied at a temperature between 36°C and 38°C. (Item 5) The method according to any one of items 1 to 4, wherein the ultrasonic frequency is applied at a temperature of approximately 37°C. (Item 6) The method according to any one of items 1 to 5, wherein the mammalian ECM is present at a concentration of approximately 25 mg / ml to approximately 600 mg / ml. (Item 7) The method according to any one of items 1 to 6, wherein the ultrasonic frequency is 20 kHz or greater. (Item 8) The method according to any one of items 1 to 7, wherein the ultrasonic frequency is approximately 20 kHz to approximately 100 kHz. (Item 9) The method according to any one of items 1 to 8, wherein the ultrasonic frequency is applied for at least 30 seconds. (Item 10) A method for generating an extracellular matrix (ECM) hydrogel, comprising solubilizing mammalian ECM in a liquid at a concentration of approximately 25 mg / ml to approximately 600 mg / ml for at least approximately 30 seconds at a temperature higher than approximately 37°C using ultrasound with a frequency of approximately 20 kHz to approximately 100 kHz, thereby generating a liquid-phase acoustic ECM hydrogel. (Item 11) The method according to any one of items 1 to 10, wherein the mammalian ECM is treated with ultrasound for at least about 60 seconds. (Item 12) The method according to any one of items 1 to 11, wherein the mammalian ECM is freeze-dried mammalian ECM. (Item 13) The method according to item 12, wherein the freeze-dried mammalian ECM is ground into a powder. (Item 14) The method according to any one of items 1 to 13, wherein the ECM is provided as a fragment in the range of about 10 μm to about 2000 μm. (Item 15) The method according to any one of items 1 to 13, wherein the ECM is provided as a fragment in the range of about 10 μm to about 1000 μm. (Item 16) The method according to any one of items 1 to 15, further comprising cooling the liquid-phase acoustic ECM hydrogel to a temperature of about 37°C or below, thereby generating a gel-phase acoustic ECM hydrogel. (Item 17) The method according to item 16, wherein the cooling is performed at a temperature of 4°C to 25°C. (Item 18) The method according to item 16, wherein the cooling is performed at a temperature between 4°C and less than 37°C. (Item 19) The method according to any one of items 1 to 18, wherein the ultrasonic wave has a frequency of approximately 20 kHz. (Item 20) The method according to any one of items 1 to 19, wherein the ultrasound has an amplitude of approximately 20 μm to approximately 320 μm. (Item 21) The method according to item 20, wherein the ultrasound has an amplitude of approximately 36 μm to approximately 180 μm. (Item 22) The method according to any one of items 1 to 21, comprising treating the solubilized mammalian ECM in the liquid with ultrasound for about 60 seconds to about 1 hour. (Item 23) The method according to any one of items 1 to 22, comprising treating the solubilized mammalian ECM in the liquid with the ultrasound for about 60 seconds. (Item 24) The method according to any one of items 1 to 23, comprising providing the mammalian ECM in the liquid at a concentration of 25 mg / ml to 150 mg / ml. (Item 25) The method according to any one of items 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with the ultrasound at a temperature of about 30 to 45°C. (Item 26) The method according to any one of items 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with the ultrasound at a temperature of about 35°C to 40°C. (Item 27) The method according to any one of items 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with the sonication at a temperature of about 36°C to 38°C. (Item 28) The method according to any one of items 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with the ultrasound at a temperature of about 37°C. (Item 29) The method according to any one of items 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with the ultrasound at a temperature of about 40°C. (Item 30) The method according to any one of items 1 to 29, wherein the ECM is human ECM. (Item 31) The method according to any one of items 1 to 30, wherein the liquid is phosphate-buffered saline. (Item 32) The method according to any one of items 1 to 31, wherein the ECM is a bladder ECM, a small intestine submucosal ECM, an esophageal ECM, a tracheal ECM, a liver ECM, or a dermal ECM. (Item 33) The method according to any one of items 1 to 32, wherein the ECM is porcine ECM or bovine ECM. (Item 34) The method according to any one of items 1 to 33, wherein the ECM is porcine esophageal ECM. (Item 35) The method according to any one of items 1 to 33, wherein the ECM is a pig bladder ECM. (Item 36) The method according to any one of items 1 to 35, further comprising placing the liquid phase of the acoustic ECM hydrogel into a three-dimensional mold before cooling. (Item 37) The method according to item 36, wherein the mold produces a sheet with a thickness of at least 4 microns. (Item 38) The method according to any one of items 1 to 37, wherein the acoustic ECM hydrogel is irradiated with gamma rays. (Item 39) The method according to item 38, wherein the acoustic ECM hydrogel is irradiated with gamma rays at approximately 20 kGy. (Item 40) Acoustic ECM hydrogel produced by the method described in any one of items 1-39. (Item 41) An acoustic ECM hydrogel that is thermoreversible, exists in a gel phase at temperatures below approximately 37°C, and in a liquid phase at temperatures above approximately 37°C. (Item 42) Acoustic ECM hydrogel as described in item 40 or 41, wherein the storage modulus (G') is approximately one order of magnitude greater than the loss modulus (G”). (Item 43) The acoustic ECM hydrogel according to any one of items 40 to 42, wherein the viscosity of the acoustic ECM hydrogel decreases with increasing stress at a temperature of approximately 15°C to approximately 37°C. (Item 44) An acoustic ECM hydrogel as described in any one of items 40-43, wherein the mammalian ECM hydrogel is a human ECM hydrogel. (Item 45) The acoustic ECM hydrogel described in any one of items 40-44, wherein the ECM is bladder ECM, small intestine submucosal ECM, esophageal ECM, tracheal ECM, hepatic ECM, or dermal ECM. (Item 46) The acoustic ECM hydrogel according to any one of items 40 to 45, wherein the aforementioned ECM is porcine ECM. (Item 47) Approximately 1400 Pa at 15℃ * Viscosity of s, and approximately 400 Pa at a temperature of 25°C. *An acoustic ECM hydrogel according to any one of items 40 to 46, having a viscosity of s and containing ECM at a concentration of approximately 150 mg / mL. (Item 48) Approximately 2700 Pa at 15℃ * At approximately 25°C, the pressure is around 800 Pa. * s and 600 Pa at 37℃ * An acoustic ECM hydrogel according to any one of items 40 to 47, having a storage modulus of s and containing ECM at a concentration of approximately 150 mg / mL. (Item 49) An acoustic ECM hydrogel irradiated with gamma rays, as described in any one of items 40-48. (Item 50) An acoustic ECM hydrogel described in any one of items 40 to 49, which does not contain either exogenous proteases or inactivated exogenous proteases. (Item 51) An acoustic ECM hydrogel according to any one of items 40 to 50, containing exogenous pepsin, trypsin, or hyaluronidase, or an inactivated form of exogenous pepsin, trypsin, or hyaluronidase. (Item 52) A method for increasing hemostasis in a target injury, comprising locally administering a therapeutically effective amount of the acoustic ECM hydrogel described in any one of items 40 to 51 to the injury, thereby increasing hemostasis. (Item 53) The method according to item 52, wherein the injury is a surgical wound, burn, or traumatic injury. (Item 54) The method described in item 52 or 53, wherein the subject is a human. (Item 55) The method according to any one of items 52 to 54, wherein the injury is located in the blood vessels, liver, lungs, or skin of the subject. (Item 56) The method according to any one of items 52 to 55, wherein hemostasis is induced within approximately 10 to 100 seconds after administering the acoustic ECM hydrogel to the subject. (Item 57) A method for inducing the M2 phenotype in macrophages, comprising treating macrophages with an effective amount of an acoustic ECM hydrogel described in any one of items 40 to 51, thereby inducing the M2 phenotype. (Item 58) The method described in item 57, wherein the subject is a human. (Item 59) A method of incising the muscularis propria, mucosa, and submucosa from the region of the target organ, A pharmaceutical composition comprising an acoustic ECM hydrogel as described in any one of items 40 to 51 is injected submucosa into the target organ to form a cushion between the submucosa and the underlying muscularis propria in the region of the organ. This allows the mucosa and submucosa to be incised from the underlying muscularis propria, thereby preventing inflammation in the area of ​​the target organ. A method that includes this. (Item 60) The method according to item 59, wherein the acoustic ECM hydrogel is produced from bladder ECM, small intestinal submucosal ECM, esophageal ECM, tracheal ECM, hepatic ECM, or dermal ECM. (Item 61) The method according to item 59 or item 60, wherein the ECM concentration in the acoustic ECM hydrogel is 25 mg / ml to about 600 mg / ml. (Item 62) The method according to item 59 or item 60, wherein the ECM concentration in the acoustic ECM hydrogel is 25 mg / ml to about 100 mg / ml. (Item 63) The method according to any one of items 59 to 62, wherein the acoustic ECM hydrogel is administered endoscopically or by catheter. (Item 64) The method according to any one of items 59 to 63, wherein the organ is the esophagus, stomach, colon, rectum, or small intestine. (Item 65) The method according to item 64, wherein the colon is the ascending colon, transverse colon, descending colon, or sigmoid colon. (Item 66) The method according to item 64, wherein the small intestine is the jejunum, cecum, or ileum. (Item 67) The method according to any one of items 59 to 66, including a method for incising adenocarcinoma or carcinoma from the aforementioned organ. (Item 68) The method according to item 67, wherein the organ is the stomach, small intestine, or colon. (Item 69) The method according to any one of items 64 to 65, wherein the organ is the colon. (Item 70) The method according to any one of items 64, 65, or 69, wherein the organ is the colon, and the method comprises incising a polyp or carcinoma from the colon. (Item 71) The method according to any one of items 59 to 64, wherein the organ is the esophagus, and the method comprises incising the mucosa and submucosa from the muscularis propria of the esophagus. (Item 72) The method according to item 71, wherein the subject has Barrett's esophagus. (Item 73) The method according to any one of items 59 to 72, further comprising performing an endoscopic resection procedure on the cushion to remove the incised mucosa and submucosa. (Item 74) The method according to item 73, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection. (Item 75) The method according to item 74, comprising dividing the cushion so that the acoustic ECM hydrogel is held on the muscularis propria of the organ beneath it, and the mucosa and submucosa are removed from the region of the organ. (Item 76) The method described in any one of items 59 to 75, wherein the subject is a human. (Item 77) The method according to any one of items 59 to 76, wherein the aforementioned organ is located in the gastrointestinal tract.

Claims

1. A method for producing an extracellular matrix (ECM) hydrogel, comprising solubilizing mammalian ECM in a liquid using an ultrasonic frequency to produce a liquid-phase acoustic ECM hydrogel.

2. The method according to claim 1, wherein the ultrasonic frequency is applied at a temperature between 30°C and 43°C.

3. The method according to claim 1 or 2, wherein the ultrasonic frequency is applied at a temperature between 35°C and 40°C.

4. The method according to any one of claims 1 to 3, wherein the ultrasonic frequency is applied at a temperature between 36°C and 38°C.

5. The method according to any one of claims 1 to 4, wherein the ultrasonic frequency is applied at a temperature of approximately 37°C.

6. The method according to any one of claims 1 to 5, wherein the mammalian ECM is present at a concentration of about 25 mg / ml to about 600 mg / ml.

7. The method according to any one of claims 1 to 6, wherein the ultrasonic frequency is 20 kHz or greater.

8. The method according to any one of claims 1 to 7, wherein the ultrasonic frequency is from about 20 kHz to about 100 kHz.

9. The method according to any one of claims 1 to 8, wherein the ultrasonic frequency is applied for at least 30 seconds.

10. A method for producing an extracellular matrix (ECM) hydrogel, comprising solubilizing mammalian ECM in a liquid at a concentration of about 25 mg / ml to about 600 mg / ml for at least about 30 seconds at a temperature higher than about 37°C using ultrasound with a frequency of about 20 kHz to about 100 kHz, thereby producing a liquid-phase acoustic ECM hydrogel.

11. The method according to any one of claims 1 to 10, wherein the mammalian ECM is treated with ultrasound for at least about 60 seconds.

12. The method according to any one of claims 1 to 11, wherein the mammalian ECM is freeze-dried mammalian ECM.

13. The method according to claim 12, wherein the freeze-dried mammalian ECM is ground into a powder.

14. The method according to any one of claims 1 to 13, wherein the ECM is provided as a fragment in the range of about 10 μm to about 2000 μm.

15. The method according to any one of claims 1 to 13, wherein the ECM is provided as a fragment in the range of about 10 μm to about 1000 μm.

16. The method according to any one of claims 1 to 15, further comprising cooling the liquid phase acoustic ECM hydrogel to a temperature of about 37°C or less, thereby generating the gel phase acoustic ECM hydrogel.

17. The method according to claim 16, wherein the cooling is performed at a temperature of 4°C to 25°C.

18. The method according to claim 16, wherein the cooling is performed at a temperature between 4°C and less than 37°C.

19. The method according to any one of claims 1 to 18, wherein the ultrasonic wave has a frequency of about 20 kHz.

20. The method according to any one of claims 1 to 19, wherein the ultrasonic wave has an amplitude of about 20 μm to about 320 μm.

21. The method according to claim 20, wherein the ultrasonic wave has an amplitude of about 36 μm to about 180 μm.

22. The method according to any one of claims 1 to 21, comprising treating the solubilized mammalian ECM in the liquid with ultrasound for about 60 seconds to about 1 hour.

23. The method according to any one of claims 1 to 22, comprising treating the solubilized mammalian ECM in the liquid with ultrasound for about 60 seconds.

24. The method according to any one of claims 1 to 23, comprising providing the mammalian ECM in the liquid at a concentration of 25 mg / ml to 150 mg / ml.

25. The method according to any one of claims 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 30 to 45°C.

26. The method according to any one of claims 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 35°C to 40°C.

27. ​​The method according to any one of claims 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 36°C to 38°C.

28. The method according to any one of claims 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 37°C.

29. The method according to any one of claims 1 to 24, comprising treating the solubilized mammalian ECM in the liquid with ultrasound at a temperature of about 40°C.

30. The method according to any one of claims 1 to 29, wherein the ECM is human ECM.

31. The method according to any one of claims 1 to 30, wherein the liquid is a phosphate-buffered saline solution.

32. The method according to any one of claims 1 to 31, wherein the ECM is bladder ECM, submucosal ECM of the small intestine, esophageal ECM, tracheal ECM, liver ECM, or dermal ECM.

33. The method according to any one of claims 1 to 32, wherein the ECM is porcine ECM or bovine ECM.

34. The method according to any one of claims 1 to 33, wherein the ECM is porcine esophageal ECM.

35. The method according to any one of claims 1 to 33, wherein the ECM is a pig bladder ECM.

36. The method according to any one of claims 1 to 35, further comprising placing the liquid-phase acoustic ECM hydrogel into a three-dimensional mold before cooling.

37. The method according to claim 36, wherein the mold produces a sheet with a thickness of at least 4 microns.

38. The method according to any one of claims 1 to 37, wherein the acoustic ECM hydrogel is irradiated with gamma rays.

39. The method according to claim 38, wherein the acoustic ECM hydrogel is irradiated with gamma rays at approximately 20 kGy.

40. An acoustic ECM hydrogel produced by the method described in any one of claims 1 to 39.

41. An acoustic ECM hydrogel that is thermoreversible, is in a gel phase at temperatures below about 37°C, and is in a liquid phase at temperatures above about 37°C.

42. The acoustic ECM hydrogel according to claim 40 or 41, wherein the storage modulus (G') is about an order of magnitude greater than the loss modulus (G'').

43. The acoustic ECM hydrogel according to any one of claims 40 to 42, wherein the viscosity of the acoustic ECM hydrogel decreases with increasing stress at a temperature of about 15°C to about 37°C.

44. The acoustic ECM hydrogel according to any one of claims 40 to 43, wherein the mammalian ECM hydrogel is a human ECM hydrogel.

45. The acoustic ECM hydrogel according to any one of claims 40 to 44, wherein the ECM is bladder ECM, submucosal ECM of the small intestine, esophageal ECM, tracheal ECM, liver ECM, or dermal ECM.

46. The acoustic ECM hydrogel according to any one of claims 40 to 45, wherein the ECM is porcine ECM.

47. The acoustic ECM hydrogel according to any one of claims 40 to 46, comprising ECM having 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 a concentration of about 150 mg / mL.

48. The acoustic ECM hydrogel according to any one of claims 40 to 47, comprising ECM with a storage modulus of approximately 2700 Pa*s at 15°C, approximately 800 Pa*s at 25°C, and 600 Pa*s at 37°C, and a concentration of approximately 150 mg / mL.

49. An acoustic ECM hydrogel according to any one of claims 40 to 48, which has been irradiated with gamma rays.

50. The acoustic ECM hydrogel according to any one of claims 40 to 49, which does not contain either an exogenous protease or an inactivated exogenous protease.

51. The acoustic ECM hydrogel according to any one of claims 40 to 50, comprising exogenous pepsin, trypsin, or hyaluronidase, or an inactivated form of exogenous pepsin, trypsin, or hyaluronidase.

52. A method for increasing hemostasis in a target injury, comprising locally administering a therapeutically effective amount of the acoustic ECM hydrogel described in any one of claims 40 to 51 to the injury, thereby increasing hemostasis.

53. The method according to claim 52, wherein the injury is a surgical wound, a burn, or a traumatic injury.

54. The method according to claim 52 or 53, wherein the subject is a human.

55. The method according to any one of claims 52 to 54, wherein the injury is located in the blood vessels, liver, lungs, or skin of the subject.

56. The method according to any one of claims 52 to 55, wherein hemostasis is induced within about 10 to about 100 seconds after administering the acoustic ECM hydrogel to the subject.

57. A method for inducing the M2 phenotype in macrophages, comprising treating macrophages with an effective amount of the acoustic ECM hydrogel described in any one of claims 40 to 51, thereby inducing the M2 phenotype.

58. The method according to claim 57, wherein the subject is a human.

59. A method for incising the mucosa and submucosa from the muscularis propria of a target organ, A pharmaceutical composition comprising the acoustic ECM hydrogel described in any one of claims 40 to 51 is injected submucosa into the target organ to form a cushion between the submucosa and the underlying muscularis propria in the region of the organ. This allows the mucosa and submucosa to be incised from the underlying muscularis propria, thereby preventing inflammation in the area of ​​the target organ. A method that includes this.

60. The method according to claim 59, wherein the acoustic ECM hydrogel is produced from bladder ECM, submucosal ECM of the small intestine, esophageal ECM, tracheal ECM, liver ECM, or dermal ECM.

61. The method according to claim 59 or claim 60, wherein the ECM concentration in the acoustic ECM hydrogel is 25 mg / ml to about 600 mg / ml.

62. The method according to claim 59 or claim 60, wherein the ECM concentration in the acoustic ECM hydrogel is 25 mg / ml to about 100 mg / ml.

63. The method according to any one of claims 59 to 62, wherein the acoustic ECM hydrogel is administered endoscopically or by catheter.

64. The method according to any one of claims 59 to 63, wherein the organ is the esophagus, stomach, colon, rectum, or small intestine.

65. The method according to claim 64, wherein the colon is the ascending colon, transverse colon, descending colon, or sigmoid colon.

66. The method according to claim 64, wherein the small intestine is the jejunum, cecum, or ileum.

67. The method according to any one of claims 59 to 66, comprising a method for incising an adenocarcinoma or carcinoma from the organ.

68. The method according to claim 67, wherein the organ is the stomach, small intestine, or colon.

69. The method according to any one of claims 64 to 65, wherein the organ is the colon.

70. The method according to any one of claims 64, 65, or 69, wherein the organ is the colon, and the method comprises incising a polyp or carcinoma from the colon.

71. The method according to any one of claims 59 to 64, wherein the organ is the esophagus, and the method comprises incising the mucosa and submucosa from the muscularis propria of the esophagus.

72. The method according to claim 71, wherein the subject has a Barrett's esophagus.

73. The method according to any one of claims 59 to 72, further comprising performing an endoscopic resection procedure on the cushion to remove the incised mucosa and submucosa.

74. The method according to claim 73, wherein the resection procedure is endoscopic mucosal resection or endoscopic submucosal dissection.

75. The method according to claim 74, comprising dividing the cushion so that the acoustic ECM hydrogel is held on the muscularis propria of the organ beneath it, and the mucosa and submucosa are removed from the region of the organ.

76. The method according to any one of claims 59 to 75, wherein the subject is a human.

77. The method according to any one of claims 59 to 76, wherein the organ is located in the gastrointestinal tract.

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