Biofabricated vaginal extracellular matrix hydrogel

Abstract

OF THE INVENTION Provided herein is a method of preparing an extracellular matrix (ECM) material, including incubating vaginal tissue in a protease, incubating the vaginal tissue in a solution including at least one detergent, incubating the vaginal tissue in a composition including at least one disinfecting agent, thereby producing a decellularized ECM material, lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material, comminuting the lyophilized ECM material, partially or completely solubilizing the comminuted, lyophilized ECM material with an acid protease to produce solubilized ECM, lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

Description

BIOFABRICATED VAGINAL EXTRACELLULAR MATRIX HYDROGELCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 63 / 603,929, filed November 29, 2023, the disclosure of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention

[0002] Provided herein are cell scaffolds useful for generating therapeutic compositions, for treating various conditions, and as vehicles for delivery of therapeutic compositions.Description of Related Art

[0003] The vagina is a dynamic, mechanically robust organ that remains understudied in human and animal biology. This has impaired investigations into novel, regenerative medicinebased strategies for restoring vaginal integrity for maintenance of sexual health and pelvic organ function. Extirpative surgeries for pelvic organ cancers, radiation treatment, and congenital anomalies can result in loss of all or a significant portion of the vagina. Ovariectomy, menopause, aging, chemotherapy, and radiation in women who cannot use local estrogen often lead to a vagina that is foreshortened, contracted, thinned, and painful. Trauma from vaginal birth and age predispose to loss of vaginal support to the pelvic organs resulting in their descent into the vagina - a condition referred to as pelvic organ prolapse (POP). To date, devices to biofabricate the vagina or restore vaginal structure and function fall short. Indeed, there are no robust regenerative strategies to maintain vaginal health or to restore structure and function. Further, current surgical strategies to repair these conditions are prone to complications and are limited in their optimization through research which, to date, has required expensive animal models and laboratory personnel. Finally, there are no robust three- dimensional culture models to investigate vaginal pathobiology. Accordingly, there is a need in the art for new materials to address these known shortcomings.SUMMARY OF THE INVENTION

[0004] Provided herein is a method of preparing an extracellular matrix (ECM) material, including incubating vaginal tissue in a protease, incubating the vaginal tissue in a solution including at least one detergent, incubating the vaginal tissue in a composition including at least one disinfecting agent, thereby producing a decellularized ECM material, lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material, comminuting thelyophilized ECM material, partially or completely solubilizing the comminuted, lyophilized ECM material with an acid protease to produce solubilized ECM, lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0005] Also provided herein is a method of preparing an extracellular matrix (ECM) material, including exposing vaginal tissue to at least one freeze-thaw cycle, disinfecting the vaginal tissue, optionally with at least one antibiotic and / or antimycotic composition, incubating the vaginal tissue in a solution including at least one detergent, thereby producing a decellularized ECM material, lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material, comminuting the lyophilized ECM material, partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM, and lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0006] Also provided herein is a method of preparing an extracellular matrix (ECM) material, including exposing vaginal tissue to at least one freeze-thaw cycle, disinfecting the tissue, optionally with at least one antibiotic and / or antimycotic composition, thereby producing a decellularized ECM material, lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material, comminuting the lyophilized ECM material, partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM, and lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0007] Also provided herein is an ECM material prepared by any of the aforementioned methods.

[0008] Also provided herein is a method of treating a defect or condition in a patient using the ECM material prepared by any of the aforementioned methods.

[0009] Further non-limiting embodiments are set forth in the following numbered clauses:

[0010] 1. A method of treating a condition in a patient, comprising administering to the patient, an extracellular matrix (ECM) composition comprising decellularized, acid-protease- digested ECM derived from vaginal tissue.

[0011] 2. The method of clause 1, wherein the vaginal tissue is human, bovine, ovine, or porcine.

[0012] 3. The method of clause 1 or clause 2, wherein the acid-protease-digested ECM is not dialyzed or chemically crosslinked.

[0013] 4. The method of any of clauses 1-3, wherein the composition forms a gel when warmed to 37°C.

[0014] 5. The method of any of clauses 1-4, wherein the ECM composition is applied to a mesh or graft that is implanted in the patient.

[0015] 6. The method of any of clauses 1-5, wherein the mesh or graft comprises an inorganic material.

[0016] 7. The method of any of clauses 1-6, wherein the ECM composition is coated onto a surface of the mesh or graft.

[0017] 8. The method of any of clauses 1-7, wherein the ECM composition is seeded with one or more cells.

[0018] 9. The method of any of clauses 1-8, wherein the one or more cells comprise one or more fibroblasts.

[0019] 10. The method of any of clause 1-9, wherein the ECM composition comprises one or more therapeutic agents.

[0020] 11. An extracellular matrix (ECM) gel comprising acid-protease-digested ECM derived from vaginal tissue and one or more cells dispersed therein.

[0021] 12. A method of preparing an extracellular matrix (ECM) material, comprising: a. incubating vaginal tissue in a protease; b. incubating the vaginal tissue in a solution comprising at least one detergent; c. incubating the vaginal tissue in a composition comprising at least one disinfecting agent, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e. comminuting the lyophilized ECM material; f. partially or completely solubilizing the comminuted, lyophilized ECM material with an acid protease to produce solubilized ECM; and g. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0022] 13. A method of preparing an extracellular matrix (ECM) material, comprising: a. exposing vaginal tissue to at least one freeze-thaw cycle; b. disinfecting the vaginal tissue, optionally with at least one antibiotic and / or antimycotic composition; c. incubating the vaginal tissue in a solution comprising at least one detergent, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e. comminuting the lyophilized ECM material; f. partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM; and g. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0023] 14. The method of clause 12 or clause 13, further comprising, after lyophilizing the solubilized ECM, neutralizing the lyophilized solubilized ECM to produce an ECM pre-gel.

[0024] 15. The method of any of clauses 12-14, wherein: the detergent is a zwiterionic detergent and / or an anionic detergent, optionally CHAPs, SDS, sodium deoxycholate, and / or Triton-X-100; and / or the disinfecting agent is ethanol and / or peracetic acid.

[0025] 16. The method of any of clauses 12-15, further comprising gelling the ECM pregel at a temperature at which the ECM pre-gel gels to produce an ECM gel.

[0026] 17. The method of any of clauses 12-16, wherein the decellularized ECM material is not completely digested with the acid protease, producing an ECM pre-gel that is able to gel at 37°C comprising undigested decellularized ECM particles.

[0027] 18. The method of any of clauses 12-17, further comprising including one or more washing steps from prior to the step of comminuting the lyophilized ECM material.

[0028] 19. The method of any of clauses 12-18, wherein the one or more washing steps comprises washing the tissue or material with phosphate-buffered saline, saline, and / or water.

[0029] 20. The method of any of clauses 12-19, wherein the ECM material is prepared without a dialysis step or a crosslinking step.

[0030] 21. The method of any of clauses 12-20, wherein the acid protease is pepsin and / or trypsin.

[0031] 22. The method of any of clauses 12-21, wherein the lyophilized ECM material is solubilized with an acid protease in a solution having a pH of from 1 to 4, from 1 to 2, or 2.0 ± 0.3.

[0032] 23. The method of any of clauses 12-22, comprising dispersing the ECM material in a natural or a synthetic polymer composition.

[0033] 24. The method of any of clauses 12-23, wherein the natural or a synthetic polymer composition is one or more of: a second ECM material, fibrin, collagen, polyester (PE), polyurethane (PU), poly(ester urethane) urea (PEUU), poly(ether ester urethane) urea (PEEUU), poly (ester carbonate urethane)urea (PECUU), poly (carbonate urethane)urea (PCUU) copolymer, polyolefin (poly alkene), polycarbonate, poly anhydride, poly ether, polyurea, polyurethane, polyketone, and fluoropolymer.

[0034] 25. The method of any of clauses 12-24, wherein the ECM material is mixed with the natural or synthetic polymer composition prior to or during gelation of the ECM material.

[0035] 26. The method of any of clauses 12-25, wherein the pre-gel is mixed with fibrin and fibrinogen and is gelled while the fibrin is cross-linked with the fibrinogen.

[0036] 27. An ECM composition comprising decellularized, acid-protease-digested vaginal tissue, having a pH of from 6.8 to 7.8.

[0037] 28. The ECM composition of clause 27, wherein the composition is a gel and as compared to acid-protease-digested porcine small intestine submucosa, the gel comprises longer fibers and at least 50% lower FGF-1 and / or FGF-2 content, and optionally has increased HB-EGF (Heparin Binding EGF Eike Growth Factor) content and / or lower content of one or more of Angiopoietin 2; Endostatin; IGFBP1 (Insulin Like Growth Factor Binding Protein 1); PTX3 (Pentraxin 3); Prolactin; Serpin B5; and / or TIMP4 (TIMP Metallopeptidase Inhibitor 4), and optionally has at least 50% lower FGF-1 and / or FGF-2 content, increased HB-EGF (Heparin Binding EGF Like Growth Factor) content, and lower content of Angiopoietin 2; Endostatin; IGFBP1 (Insulin Like Growth Factor Binding Protein 1); PTX3 (Pentraxin 3); Prolactin; Serpin B5; and TIMP4 (TIMP Metallopeptidase Inhibitor 4).

[0038] 29. The composition of clause 27 or clause 28, wherein the acid-protease-digested vaginal tissue is not dialyzed or chemically crosslinked.

[0039] 30. The composition of any of clauses 27-29, wherein the composition is a lyophilized powder.

[0040] 31. A method of preparing an extracellular matrix (ECM) material, comprising: a. exposing vaginal tissue to at least one freeze-thaw cycle; b. disinfecting the tissue, optionally with at least one antibiotic and / or antimycotic composition, thereby producing a decellularized ECM material; c. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; d. comminuting the lyophilized ECM material; e. partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM; and f. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

[0041] 32. The method of clause 31, further comprising neutralizing the lyophilized, solubilized ECM to produce an ECM pre-gel.

[0042] 33. The method of clause 31 or clause 32, further comprising including one or more washing steps prior to the step of comminuting the lyophilized ECM material.

[0043] 34. The method of any of clauses 31-33, wherein the one or more washing steps comprises washing the tissue or material with phosphate-buffered saline, saline, and / or water.

[0044] 35. The method of any of clauses 31-34, wherein the ECM material is prepared without a dialysis step or a crosslinking step.

[0045] 36. The method of any of clauses 31-35, wherein the acid protease is pepsin and / or trypsin.

[0046] 37. The method of any of clauses 31-36, wherein the lyophilized ECM material is solubilized with an acid protease in a solution having a pH of from 1 to 4, from 1 to 2, or 2.0 ± 0.3.

[0047] 38. The method of any of clauses 31-37, comprising dispersing the ECM material in a natural or a synthetic polymer composition.

[0048] 39. The method of any of clauses 31-38, wherein the natural or a synthetic polymer composition is one or more of: a second ECM material, fibrin, collagen, polyester (PE), polyurethane (PU), poly(ester urethane) urea (PEUU), poly(ether ester urethane) urea (PEEUU), poly (ester carbonate urethane)urea (PECUU), poly (carbonate urethane)urea (PCUU) copolymer, polyolefin (poly alkene), polycarbonate, poly anhydride, poly ether, polyurea, polyurethane, polyketone, and fluoropolymer.

[0049] 40. The method of any of clauses 31-39, wherein the ECM material is mixed with the natural or synthetic polymer composition prior to or during gelation of the ECM material.

[0050] 41. The method of any of clauses 31-40, wherein the pre-gel is mixed with fibrin and fibrinogen and is gelled while the fibrin is cross-linked with the fibrinogen.

[0051] 42. A method of preparing an extracellular matrix (ECM) material, comprising: a. incubating vaginal tissue in trypsin; b. incubating the vaginal tissue in a solution comprising Triton-X-100; c. incubating the vaginal tissue in a solution comprising sodium deoxycholate; c. incubating the vaginal tissue in a composition comprising ethanol and / or peracetic acid, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e. comminuting the lyophilized ECM material; f. partially or completely solubilizing the comminuted, lyophilized ECM material with pepsin to produce solubilized ECM; and g. lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIGS. 1A-B is a schematic diagram showing a non-limiting embodiment of a method of forming a vaginal extracellular matrix (vECM) composition as described herein;

[0053] FIG. 2 shows hematoxylin and eosin (H&E) staining of vaginal tissue decellularized by various methods, including control (non-treated) tissue (NT), physical decellularization (FT), detergent-based decellularization (SDC), and a hybrid of physical and detergent-based decellularization (FT+T);

[0054] FIGS. 3A-3B shows collagen levels of vaginal tissue decellularized by various methods, including control (non-treated) tissue (NT), physical decellularization (FT),detergent-based decellularization (SDC), and a hybrid of physical and detergent-based decellularization (FT+T) (n=20);

[0055] FIGS. 4A-4B shows glycosaminoglycan (GAG) levels of vaginal tissue decellularized by various methods, including control (non-treated) tissue (NT), physical decellularization (FT), detergent-based decellularization (SDC), and a hybrid of physical and detergent-based decellularization (FT+T) (n=20);

[0056] FIG. 5 shows schematics of vECM compositions with various concentrations of vECM according to non-limiting embodiments described herein;

[0057] FIG. 6 shows schematics of vECM compositions with various concentrations of vECM according to non-limiting embodiments described herein;

[0058] FIG. 7 shows rheometry of vECM gels according to non-limiting embodiments described herein;

[0059] FIG. 8 shows rheometry of vECM gels according to non-limiting embodiments described herein;

[0060] FIG. 9 shows cell penetration of vECM compositions with various concentrations of vECM seeded with fibroblasts according to non-limiting embodiments described herein;

[0061] FIG. 10 shows cellular viability and proliferation in vECM compositions with various concentrations of vECM seeded with fibroblasts according to non-limiting embodiments described herein;

[0062] FIG. 11 shows viability of fibroblasts in vECM compositions seeded with fibroblasts according to non-limiting embodiments described herein;

[0063] FIG. 12 shows activation and proliferation of fibroblasts in vECM compositions seeded with fibroblasts according to non-limiting embodiments described herein;

[0064] FIG. 13 shows human fibroblasts seeded throughout vECM gels at 5, 10, 15, and 20 mg / mL concentrations according to non-limiting embodiments described herein

[0065] FIG. 14 shows cell distribution in vECM gels, even out to 14 days as shown here, according to non-limiting embodiments described herein;

[0066] FIG. 15 shows a mesh coated with a vECM gel according to non-limiting embodiments described herein;

[0067] FIGS. 16A-16C show maintenance of native vECM components through decellularization and gelation;

[0068] FIGS. 17A-17C show evidence of the ability to control gelation at multiple concentrations, revealing a logarithmic relationship between vECM concentration and stiffness of hydrogel;

[0069] FIGS. 18A-18B show Scanning Electron Microscopy (SEM) images which were used to understand differences in hydrogel microstructure with vECM protein concentration;

[0070] FIGS. 19A-19B show evidence of hydrogel concentration affecting behavior of introduced cells;

[0071] FIGS. 20A-20C show vECM hydrogel as an in vitro model; evidencing human fibroblast activation, proliferation, and viability in the vECM hydrogel out to 14 days;

[0072] FIGS. 21A-22B show (A) vECM hydrogel injections localized to the vagina 24 hours after injection in mice with no off-target presence visualized and (B) mice demonstrating excellent tolerance of vECM hydrogel injections;

[0073] FIG. 22 shows vECM hydrogel as a biomaterial coating; and

[0074] FIGS. 23A-23C show vECM hydrogel as a biomaterial to assist in tissue reconstruction: vaginoplasty.DESCRIPTION OF THE INVENTION

[0075] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations, and the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of these ranges is intended as a continuous range including every value between the minimum and maximum values. For definitions provided herein, those definitions refer to word forms, cognates and grammatical variants of those words or phrases. As used herein “a” and “an” refer to one or more.

[0076] As used herein, the term “patient” or “subject” refers to members of the animal kingdom including but not limited to human beings and “mammal” refers to all mammals, including, but not limited to human beings.

[0077] As used herein, the “treatment” or “treating” of a condition or defect means administration to a patient by any suitable dosage regimen, procedure and / or administration route of a composition, device or structure with the object of achieving a desirable clinical / medical end-point, including attracting progenitor cells, correcting a defect, etc.

[0078] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, are open-ended and do not exclude the presence of other elements not identified. In contrast, the term “consisting of’ and variations thereof is intended to be closed-ended, and excludes additional elements in anything but trace amounts.

[0079] As used herein, the terms “extracellular matrix” and “ECM” refer to a natural scaffolding for cell growth. ECM is a complex mixture of structural and non- structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. In mammals, ECM often comprises about 90% collagen, in its various forms. The composition and structure of ECMs vary depending on the source of the tissue. For example, small intestine submucosa (SIS), urinary bladder matrix (UBM), liver stroma ECM, and dermal ECM each differ in their overall structure and composition due to the unique cellular niche needed for each tissue. In this disclosure, the terms “ECM” and “vaginal ECM” (“vECM”) may be used interchangeably.

[0080] As used herein, the term “derive” and any other word forms or cognates thereof, such as, without limitation, “derived” and “derives”, refers to a component or components obtained from any stated source by any useful method. For example and without limitation, generically, an ECM-derived gel refers to a gel comprised of components of ECM obtained from any tissue by any number of methods known in the art for isolating ECM. In another example, mammalian tissue-derived ECM refers to ECM comprised of components of a particular mammalian tissue obtained from a mammal by any useful method.

[0081] Provided here are scaffolds useful for reparative / restorative treatments, for example as an injectable compositions in the context of vaginal injury (e.g., birth trauma), vaginal fistula repair, and / or aging / menopause induced degeneration, as a carrier vehicle for introduction of cell populations into the vagina (e.g., stem cells), as a coating of polymeric meshes or membranes in the repair pelvic organ prolapse to enhance vaginal integration (also in vaginal biofabrication, for example in vaginal agenesis, dysgenesis, and / or in post-pelvic cancer surgery), as a research tool for testing the fibroblast response to a vaginal repair devices or conditions / diseases impacting the vagina, and / or as a “vagina in a dish” to improve understanding of the pathophysiology of diseases that impact the vagina. In non-limiting embodiments, such a model may be useful for measuring fibroblast to myofibroblast transition in response to repair materials. In non-limiting embodiments, the scaffolds described herein may be useful for supplementing vaginoplasty, for example in gender affirmation.

[0082] The methods described herein involve preparation of an ECM material and / or an ECM gel (for example as shown in FIGS. 1 and 2), which may be used as a scaffold, or for any purpose described herein. In non-limiting embodiments, the ECM gel is reverse gelling, or can be said to exhibit reverse thermal gelation, in that it forms a gel upon an increase in temperature. As the temperature rises above a certain temperature in a reverse gel, a hydrogel is formed. The general concept of reverse gelation of polymers and, e.g., its relation to lowercritical solution temperature (LCST) are broadly known in the chemical arts. The ECM compositions described herein are prepared, for example, from decellularized or devitalized, intact ECM as described below. An ECM gel may prepared by digestion of the ECM material with a protease, such as an acid protease, neutralization of the material to form a pre-gel, which may be in the form of a solution, and then raising the temperature of the pre-gel above a gelation temperature, for example the LCST of the pre-gel, to cause the pre-gel to gel. As used herein, the term “gel” includes hydrogels. The transition temperature for acid-protease-digested from solution to gel is typically within the range of from 10°C to 40°C and any increments or ranges therebetween, for example from 20°C to 35°C. For example, the pre-gel can be warmed to 37°C to form a hydrogel.

[0083] Tissue for preparation of ECM material, ECM-derived pre-gel solutions, and gels as described herein may be harvested in any useful manner. According to various non-limiting embodiments, the ECM materials described herein are prepared from vaginal tissue. For example and without limitation, the ECM material may be prepared from harvested porcine vaginal tissue, and in another, from human vaginal tissue. In non-limiting embodiments, the harvested vaginal tissue is frozen prior to processing.

[0084] Following tissue retrieval and / or isolation, in non-limiting embodiments inflammatory cellular contents are removed via decellularization. In non-limiting embodiments, decellularization may involve use of detergents, disinfectants, physical forms of decellularization (e.g., freeze-thaw cycles and / or cycles in hypotonic / hypertonic solutions), or any combination thereof. In non-limiting embodiments, decellularization is performed with strong proteases and / or detergents to produce vaginal ECM (vECM), and freeze drying and grinding the remaining vECM into a fine powder to provide a precursor material. This precursor material may be added to an acid protease digestion solution (e.g., pepsin and / or trypsin), which dissolves the vECM into its constituent protein chains to form a digest solution, which primes them for future reassembly into a gel structure. In non-limiting embodiments, following digestion with an acid protease, the digest solution is lyophilized once again. Without wishing to be bound by the theory, it is believed that this second lyophilization allows for a much higher concentration of ECM to be utilized in an ECM-containing composition, and, thus, it is to be understood that the processes disclosed herein, while exemplified for vECM, may be applicable to ECM from any source, including, without limitation, ovarian tissue, uterine tissue, small intestinal submucosa (SIS), cartilage, fallopian tube, skin, penile tissue, scrotal tissue, cardiac tissue, lung, kidney, esophagus, stomach, bladder, nerves / neurons, colon tissue, bone, ligament, tendon, adipose tissue, muscle, and the like known to those ofskill in the art. In non-limiting embodiments, removal of cellular contents may be achieved using freeze-thaw cycles, use of hypotonic solutions, antibiotic and / or antimycotic treatment, and any combination thereof. In non-limiting embodiments, decellularization may be accomplished with a combination of physical steps and chemical steps as disclosed herein (e.g., one or more freeze-that cycles, treatment with an antibiotic and / or anti mycotic composition, and / or treatment with a detergent, such as Triton-X-100).

[0085] In non-limiting embodiments, removal of cellular contents may include treatment with detergent. Suitable detergents for use in decellularization are known to those of skill in the art, and may include, without limitation, a zwitterionic detergent, such as CHAPS or betaines (any neutral compound having both positive and negative charges), and includes as a class detergents / surfactants such as l-dodecanoyl-sn-glycero-3-phosphocholine, 3-(4-tert-butyl-l- pyridinio)-l -propanesulfonate, 3-(N,N-dimethylalkylammonio)propanesulfonate, where alkyl is typically a linear, aliphatic hydrocarbon, such as a linear C6-22 saturated hydrocarbon, 3-(l- pyridinio)-l -propanesulfonate, surfactin, and other, as are broadly-available from commercial sources, such as Sigma-Aldrich. Anionic detergents are any useful detergents comprising a negative charge, such as, without limitation, alkylbenzene sulfonates, bile acids such as deoxycholic acid, and organosulfates, such as sodium dodecyl sulfate (SDS). Alternatives to trypsin-EDTA are known, and other enzymes for cell detachment and tissue dissociation, as are available commercially, such as collagenase, hyaluronidase, elastase, papain, protease Type XIV, alone or in combination, optionally with trypsin, for example from Sigma- Aldrich (e.g., Accutase®), and optionally chelating agents other than EDTA may be used to equal effect.

[0086] In non-limiting embodiments, removal of cellular contents may include a combination of one or more of the foregoing, for example freeze-that cycles, treatment with an antibiotic / antimycotic composition, and a detergent (e.g., 3% Triton-X-100). In non-limiting embodiments, tissue is obtained through a punch biopsy, followed by decellularization (e.g., one or more of the decellularization methods described herein), followed by lyophilization and milling, to provide a powdered vECM. In non-limiting embodiments, the powdered vECM is subject to protease digestion (e.g., pepsin digestion and or trypsin digestion), to provide a pregel material. The pre-gel material may be neutralized to provide a gel.

[0087] With continuing reference to decellularization of vaginal tissue, as a first step, fresh vaginal tissue is isolated. Using any method, such as by use of forceps or scissors, or by any automated mechanical process. The vaginal tissue may then be frozen and thawed. Next, the material may be incubated in a zwitterionic detergent and is typically washed. Washing may be done using saline, phosphate-buffered saline (PBS) and / or water, or other solvents, such asalcohol as is appropriate. The material may then incubated in a trypsin-EDTA or an equivalent for dissociating cells and tissue, typically followed by washing. Next, the material may be incubated in an anionic detergent, typically followed by washing. The material may subsequently be disinfected, for example by treatment with peracetic acid, and may then be washed. The material may then be dried, e.g. by lyophilization, and comminuted. In its dry state, the materials are optionally sterilized. In non-limiting embodiments, any of the foregoing decellularization steps may be used in any order and any combination.

[0088] Decellularized ECM can be dried, either lyophilized (freeze-dried) or air dried. The ECM composition is optionally comminuted at some point, for example prior to acid protease digestion in preparation of an ECM gel, for example prior to or after drying. The comminuted ECM can also be further processed into a powdered form by methods, for example and without limitation, such as grinding or milling in a frozen or freeze-dried state. As used herein, the term “comminute” and any other word forms or cognates thereof, such as, without limitation, “comminution” and “comminuting”, refers to the process of reducing larger particles, e.g., of dried ECM, into smaller particles, including, without limitation, by tearing, grinding, blending, shredding, slicing, milling, cutting, shredding, shearing, and pulverizing. ECM can be comminuted while in any form, including, but not limited to, hydrated forms, frozen, air-dried, lyophilized, powdered, sheet-form.

[0089] The dry, comminuted material may be rehydrated, for example in an acid, such as HC1, -pH <4.0, from about 1 to about 4, e.g. pH 1 to 2, for example about 2.0 ± 0.3, and may be digested with an acid protease, such as trypsin and / or pepsin, maintaining the pH of the solution at within the active range for the protease, e.g., < 4.0, from about 1 to about 4, from about 1 to about 2, e.g., about 2.0 ± 0.3, all values and subranges therebetween included. Digestion may be partial or complete. Partial digestion may be accomplished by use of shortened acid protease digestion times, use of lower amounts of acid protease in the reaction, and / or by digestion above the optimal pH for the acid protease. Complete digestion may typically be accomplished at an optimal pH for the acid protease, for example at pH of 2.5 or less, for example 2.0 ± 0.3.

[0090] To form a gel, the acidic solution may be neutralized, e.g. to pH 6.8 to 7.8, to form a pre-gel solution, and the solution may be incubated at a higher temperature, such as at room temperature (20°C-25°C) or 37°C (e.g., from 20° to 50°C, from 30° to 45°C, from 35° to 42°C, or at 37°C ± 5°C, 4°C, 3°C, 2°C, or 1°C) to form a gel, all values and subranges therebetween included. Prior to, during or after gelation, the pre-gel solution can be sprayed, coated, mixed, layered, poured, injected or otherwise deposited on a substrate or into a substrate, such as apolymer, a ceramic, a metal, a tissue (ex vivo, or in vivo), a different devitalized tissue product, such as a sheet of SIS ECM, a non-woven material, a suture, or any other medically-useful material. In non-limiting embodiments, the acid protease digestion is incomplete, but complete enough to produce a gel, leaving small particles of undigested ECM material within the resultant gel, which would be digested in situ during use of the composition - resulting in delayed release of therapeutic compositions thereof.

[0091] In order to prepare solubilized ECM tissue, ECM, for example comminuted ECM, may be digested with an acid protease in an acidic solution to form a digest solution. As used herein, the term “acid protease” refers to an enzyme that cleaves peptide bonds, wherein the enzyme has increased activity of cleaving peptide bonds in an acidic pH. For example and without limitation, acid proteases include pepsin and trypsin and mixtures thereof.

[0092] As an example, the digest solution of ECM may be kept at a constant stir for a certain amount of time at room temperature. In non-limiting embodiments, the pH is maintained at less than pH 4.0 or at pH 2.0 ± 0.3 during acid protease digestion of the vaginal tissue as described herein. The ECM digest can be used immediately or can be stored at -20°C or frozen at, for example and without limitation, -20°C or -80°C. In non-limiting embodiments, the ECM digest is snap frozen in liquid nitrogen. To form a “pre-gel” solution, the pH of the digest solution is raised to a pH between 6.8 and 7.8. The pH can be raised by adding one or more of a base or an isotonic buffered solution, for example and without limitation, NaOH or PBS at pH 7.4. The method optionally does not include a dialysis step prior to gelation, yielding a more-complete ECM-like matrix that typically gels at 37 °C more slowly than comparable collagen or dialyzed ECM preparations. The gel therefore retains more of the qualities of native ECM due to retention of many native soluble factors, such as, without limitation, cytokines. These factors contribute to chemoattraction of cells and proper rearrangement of tissue at the site of injury, rather than a fibrotic response that leads to unwanted scarring. In non-limiting embodiments, the ECM is dialyzed prior to gelation to remove certain soluble components.

[0093] As used herein, the term “isotonic buffered solution” refers to a solution that is buffered to a pH between 6.8 and 7.8, e.g., pH 7.4, and that has a balanced concentration of salts to promote an isotonic environment. As used herein, the term “base” refers to any compound or a solution of a compound with a pH greater than 7. For example and without limitation, the base is an alkaline hydroxide or an aqueous solution of an alkaline hydroxide. In non-limiting embodiments, the base is NaOH, or NaOH in PBS. This “pre-gel” solution can, at that point be incubated at a suitably warm temperature, for example and without limitation, at about 37°C, to gel.

[0094] In the method of preparing an ECM gel, the ECM may be partially or completely digested with the acid protease, such as trypsin and / or pepsin. The digested ECM may then be neutralized to a pH of 6.8-7.8, e.g., 7.2-7.6, or 7.4 and the neutralized and digested ECM material may be gelled by incubation at a temperature at which the material gels, e.g., at a temperature above 20, 25, 30, or 35°C, such as at 37°. The degree of digestion can be determined by comparison on a gel, or by ascertaining the degree of degradation of hyaluronic acid, for example by Western blot (anti-hyaluronic acid antibodies are commercially-available from multiple sources) or chromatographic methods, as are broadly known. For example in a partial digestion, hyaluronic acid is digested less than 50%, 40%, 30%, 25%, 20% or 10%.

[0095] Therefore, according to non-limiting embodiments described herein, an ECM composition is provided comprising acid-protease-digested vaginal tissue, having a pH of from 6.8 to 7.8. In non-limiting embodiments, the acid-protease-digested vaginal tissue is not dialyzed or chemically crosslinked - meaning at no stage during the processing of intact tissue to produce acid-protease-digested vaginal tissue has the material been dialyzed or cross-linked by addition of a chemical cross-linking agent, as is common in the production of certain devitalized ECM materials.

[0096] In non-limiting embodiments, vaginal tissue is decellularized using a solution including an acid protease (e.g. trypsin, e.g., 0.02% trypsin), a detergent (e.g., Triton-X-100, such as 3% Triton, deoxycholate, such as 4% sodium deoxy cholate), ethanol (e.g., 4% ethanol), and an antimicrobial composition (e.g., 0.26% peracetic acid). Decellularization may be achieved through use of the aforementioned compositions (acid protease, detergent, and disinfectant) applied to the tissue in a single mixed composition, or sequentially.

[0097] In non-limiting embodiments, vaginal tissue is decellularized by incubation in trypsin (e.g., 0.02% trypsin), followed by a wash, followed by incubation in Triton-X-100 (e.g., 3% Triton-X-100), followed by a wash, followed by incubation in sodium deoxycholate (e.g., 4% sodium deoxy cholate), followed by a wash, followed by incubation in ethanol and peracetic acid (e.g., 4% ethanol and 0.26% peracetic acid). In non-limiting embodiments, the washes are conducted with any known washing solution, such as saline, PBS, and / or water. In nonlimiting embodiments, the decellularized tissue is comminuted and / or milled, and lyophilized a first time. Following a first lyophilization, the decellularized, comminuted / milled tissue is digested with pepsin, followed, in non-limiting embodiments, by a second lyophilization. This digested, decellularized, dried composition of vECM may then be packaged and sold in a kit (for example, with one or more neutralizing agents). In non-limiting embodiments, vaginal tissue is decellularized by incubation in trypsin (e.g., 0.02% trypsin) for about 1 hour, followedby a detergent (e.g., 3% Triton) for about 1 hour, followed by another detergent (e.g., 4% sodium deoxycholate) for about two hours, followed by incubation in an alcohol (e.g., 4% ethanol) and an antimicrobial composition (e.g., 0.26% peracetic acid) for about 1 hour. In non-limiting embodiments, incubation in sodium deoxy cholate is followed by incubation in ethanol (e.g., 4% ethanol), followed by incubation in an antimicrobial composition (e.g., peracetic acid), each for an hour (FIG. 1).

[0098] In use, the ECM gel can be injected, sprayed, painted, poured, or otherwise applied to a surface of a tissue, e.g., vaginal tissue. Depending on the final use of the product, the composition may be applied or administered in a variety of ways, either as a dry, e.g., lyophilized powder, a solution, a gel, a foam, etc.

[0099] In non-limiting embodiments, the composition is cell-free, meaning the composition comprises no living cells, and is therefore sterile, and is optionally sterilized or disinfected. The composition can be terminally sterilized, for example by sterilization by, for example and without limitation, exposure to ethylene oxide (EtO) gas, gamma irradiation, or electron beam radiation, and in one embodiment when in a dried or lyophilized state (see, e.g., WO 2015 / 143310, incorporated herein by reference for its technical disclosure of methods of terminally- sterilizing ECM gels). The composition may be disinfected with peracetic acid, as described herein.

[0100] The composition can be administered by itself, or with a device or composition. For example, the composition can be absorbed into, adsorbed onto, mixed into, or otherwise coadministered with a cell-growth scaffold, such as an isotropic or anisotropic mass of fibers of synthetic and / or natural polymer(s), such as an electrodeposited, wet or dry spun, 3D printed, molded, or otherwise formed polymeric structure prepared from biocompatible polymeric materials, as are broadly known in the regenerative medical field, such as collagen, polyester (PE), polyurethane (PU), poly(ester urethane) urea (PEUU), poly(ether ester urethane) urea (PEEUU), poly (ester carbonate urethane)urea (PECUU), and poly (carbonate urethane)urea (PCUU) copolymers, and other suitable polymeric materials, such as are disclosed, for example and without limitation in U.S. Patent Nos. 8,535,719; 8,673,295; 8,889,791; 8,974,542 and 9,023,972, the contents of which are incorporated herein by reference in their entirety.

[0101] Additional non-limiting examples of useful polymer compositions for use in the compositions described herein include: polyolefin (polyalkene), polycarbonate, polyanhydride, poly ether, polyurea, polyurethane, poly ketone, and fluoropolymers. In non-limiting embodiments, the polymer composition is bioerodible. Non-limiting examples of biocompatible, bioerodible, elastomeric (co)polymer compositions including PEUU, PEEUU,PECUU, and PCUU. Other useful (co)polymers include, without limitation: polymers comprising monomers of alpha-hydroxy acids; polylactides, such as poly(lactide-co- glycolide), poly(L-lactide-co-caprolactone), polyglycolic acid, poly(dl-lactide-co-glycolide), poly(l-lactide-co-dl-lactide); other polyesters including polyhydroxybutyrate, poly hydroxy valerate, polydioxanone, and polyglactin; poly lactones including polycaprolactone, polyglyconate, poly(glycolide-co-trimethylene carbonate), poly(glycolide- co-trimethylene carbonate-co-dioxanone). Useful polymer compositions for use in the compositions described herein may also include acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), polycarbonate (PC), poly ether ester ketone (PEEK), polyetherimide (ULTEM) and thermoplastic elastomers (TPE), and like polymers, for example those utilized in ink jet printing.

[0102] The compositions described herein also can be mixed into polymeric compositions prior to or along with deposition of polymeric fibers or formation of structures. Alternatively, where the ECM product is not formed into a gel, ECM gel and / or synthetic polymers may be absorbed into, adsorbed onto or otherwise combined with the ECM product. In non-limiting embodiments, a composition as described herein is applied to and delivered from an ECM material, such as any commercial ECM material, such as those described herein.

[0103] In non-limiting embodiments, the composition is utilized, in vitro, for modelling cellular response to biomedical materials, such as meshes, such as polypropylene meshes. In non-limiting embodiments, the composition is utilized for embedded foreign bodies, and / or for injection into microfluidic devices.

[0104] Hydrogels may be fabricated in any useful sizes, including, without limitation, from about 4 mm to about 35 mm wide, from about 4 mm wide to about 13 mm wide, from about 13 mm wide to about 35 mm wide, from about 4 mm to about 5 mm wide, all values and subranges therebetween inclusive. In non-limiting embodiments, hydrogels may be fabricated with thicknesses of from about 1 mm to about 8 mm, all values and subranges therebetween inclusive. Such scaffolds may be useful for in vitro use by seeding cells throughout the hydrogel. In non-limiting embodiments, suitable cells may include fibroblasts, multipotent cells, stem cells, hydrogels, for example cast at 5 mg / mL, may be used to coat meshes, for example 1 cm x 3 cm strips of polypropylene pelvic mesh. In non-limiting embodiments, the coating may deform with the mesh as it is placed on tension. In non-limiting embodiments, a gel formed of vECM as described herein is no thicker than about 2 mm if the gel is seeded with cells prior to casting / use (e.g., in vitro or in vivo).

[0105] Likewise, the compositions described herein can be applied to or incorporated into, by any suitable method, a non-woven material, such as a mesh, a bandage, a suture, an implant, such as a ceramic, metal, or polymeric implant, for example a prosthesis, artificial or otherwise- modified vessel, a valve, an intraocular lens, a tissue transplant or implant.

[0106] As used herein, the term “coat”, and related cognates such as “coated” and “coating,” refers to a process comprising of covering an organic, inorganic, or living structure, or combinations thereof, with a composition described herein. For example and without limitation, coating of an inorganic structure with an ECM-derived gel can include methods such as pouring, embedding, layering, dipping, spraying (e.g., electrospraying), and / or printing (e.g., 3D printing). Ultrasonication may be used to aid in coating of an inorganic structure with the ECM-derived gel. As used herein, the term “ultrasonication” refers to the process of exposing ultrasonic waves typically with a frequency higher than 15 kHz and lower than 400 kHz. Organic structures include both synthetic and natural polymer compositions including devitalized tissue, proteinaceous compositions such as collagen, and synthetic polymer compositions, such as PEUU, PEEUU, PCUU, and / or PECUU, as indicated above. Living tissue may be any living tissue, whether located in situ within a patient or dissected, or not located in situ. For example, the compositions and materials described herein may be applied (in situ) to an existing tissue within the patient. In non-limiting embodiments, living tissue is treated with the described compositions, such as soaked, sprayed, and / or wrapped, prior to implantation.

[0107] In non-limiting embodiments, the composition is combined with other compositions to form a composite structure. The other compositions can be other biocompatible polymer compositions, in which the ECM gel described herein contains particles of the other biocompatible polymer, or the ECM gel is dispersed, either homogeneously or non- homogeneously (e.g., as microparticles or nanoparticles) within the other polymer. In nonlimiting embodiments, the other biocompatible polymer is a fibrin plug having gel particles of the described ECM dispersed throughout. In non-limiting embodiments, the other biocompatible polymer is a different ECM gel into which the described ECM gel is mixed either homogeneously or non-homogeneously. Other biocompatible particles include natural polymer compositions, such as, without limitation, fibrin, or synthetic polymers, such as described above.

[0108] In non-limiting embodiments, the composition is coated onto a biocompatible material, such as a metal (or alloy thereof), a polymer, and / or a ceramic composition. Nonlimiting examples of suitable metals are cobalt-chrome alloys, stainless steel alloys, titaniumalloys, tantalum alloys, titanium-tantalum alloys, which can include both non-metallic and metallic components, such as molybdenum, tantalum, niobium, zirconium, iron, manganese, chromium, cobalt, nickel aluminum and lanthanum, including without limitation, CP Ti (commercially pure titanium) of various grades or Ti-6A1-4V (90% wt. Ti, 6% wt. Al and 4% wt. V), stainless steel 316, Nitinol (Nickel-titanium alloy), titanium alloys coated with hydroxyapatite. Metals are useful due to high strength, flexibility, and biocompatibility. Metals also can be formed into complex shapes and many can withstand corrosion in the biological environments, reduce wear, and not cause damage to tissues. In non-limiting embodiments, the vECM composition disclosed herein is coated onto a mesh, for example a polypropylene mesh. In non-limiting embodiments, the material to be coated with vECM compositions as described herein, for example vECM pre-gel or gel, is pre-treated. In nonlimiting embodiments, such pre-treatments include plasma treatments, for example O2 plasma treatments. In non-limiting embodiments, a material may be coated with more than one layer of vECM composition (e.g., pre-gel or gel), and each layer may include the same or different concentrations of vECM. Without wishing to be bound by the theory, utilizing differing concentrations of vECM provides distinct stiffness, and allows for more robust modelling of diseases and regenerative constructs, both in vitro and in vivo.

[0109] In non-limiting embodiments, the composition is used for release of one or more therapeutic agents within a patient’s body and / or incorporates one or more therapeutic agents. For example, at least one therapeutic agent, in an amount effective to cause a desired response, may be added to the vECM-containing composition described herein before it is implanted in the patient or otherwise administered to the patient, for example, a therapeutic agent is added to the described compositions (e.g., vECM-containing compositions) as they are combined (e.g., prior to gelation) and / or after gelation. Generally, the therapeutic agents include any substance that can be coated on, embedded into, absorbed into, adsorbed to, or otherwise attached to or incorporated onto or into the composition or materials described herein, or incorporated into a drug product that would provide a therapeutic benefit to a patient. Nonlimiting examples of such therapeutic agents include hormones, drugs, growth factors, cytokines, cells, and the like. Each therapeutic agent may be used alone or in combination with other therapeutic agents.

[0110] In non-limiting embodiments, the therapeutic agent is a hormone and / or prohormone, such as estrogen, progestin, progesterone, growth hormone, thyroid-stimulating hormone, oxytocin, follicle-stimulating hormone, luteinizing hormone, testosterone, cortisol, prolactin, corticotropin-releasing hormone, gonadotrophin-releasing hormone, somatostatin,thyrotropin-releasing hormone, antidiuretic hormone, corticotropin, melatonin, thyroxine, triiodothyronine, reverse triiodothyronine, calcitonin, aldosterone, DHEA, epinephrine, norepinephrine, insulin, glucagon, leptin, adiponectin, plasminogen activator inhibitor- 1, angiotensin, angiotensinogen, erythropoietin, renin, vitamin D, insulin-like growth factors (IGFs), such as IGF-1, ghrelin, somatostatin, glucagon-like peptides (GEPs), such as GLP-1, and / or the like. In non-limiting embodiments, the therapeutic agent is a combination, for example combinations commonly known in birth control medications (e.g., estrogen and progestin, or progestin alone).

[0111] In non-limiting embodiments, the therapeutic agent is a growth-enhancing factor, which as utilized herein means a composition that enhances, accelerates, and / or promotes healing following trauma. In non-limiting embodiments, such growth-enhancing factors include growth factors, such as, without limitation, a neurotrophic or angiogenic factor, which optionally may be prepared using recombinant techniques. Non-limiting examples of growth factors include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), neurotrophin-3, neuro trophin-4, neurotrophin-5, pleiotrophin protein (neurite growthpromoting factor 1), midkine protein (neurite growth-promoting factor 2), brain-derived neurotrophic factor (BDNF), tumor angiogenesis factor (TAF), corticotrophin releasing factor (CRF), transforming growth factors a and P (TGF-a and TGF-P), interleukin-8 (IE-8), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins, and interferons. Commercial preparations of various growth factors, including neurotrophic and angiogenic factors, are available from R & D Systems, Minneapolis, Minnesota; Biovision, Inc, Mountain View, California; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel; and Cell Sciences®, Canton, Massachusetts.

[0112] In non-limiting embodiments, the therapeutic agent is a cytokine, for example a pro- inflammatory cytokine and / or an anti-inflammatory cytokine. In non-limiting embodiments, the therapeutic agent may be, without limitation, an interleukin, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, and / or IL-40, tumor necrosis factors (TNF), such as TNF-a, interferons (IFN), such as IFN-a, IFN-P, and / or IFN-y, colony-stimulatingfactors (CSF), such as G-CSF, chemokines, lymphokines, monokines, and the like known to those of skill in the art.

[0113] In certain non-limiting embodiments, the therapeutic agent is a drug. As used herein, the terms “drug” and “drugs” refer to any compositions having a preventative or therapeutic effect, including and without limitation, antibiotics, antivirals, antimycotics, peptides, hormones, organic molecules, steroids, NSAIDS, vitamins, supplements, factors (including growth factors), proteins, and chemoattractants.

[0114] In non-limiting embodiments, the drug is an antimicrobial agent, such as, without limitation, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione, and silver salts such as chloride, bromide, iodide and periodate. Exemplary antimicrobial agents are known to those of skill in the art.

[0115] In non-limiting embodiments, the drug is an antimycotic agent, such as clotrimazole, fluconazole, ketoconazole, posconazole, voriconazole, isavuconazole, nystatin, amphotericin B, flucytosine, echinandins, and / or micafungin. Exemplary antimycotic agents are known to those of skill in the art.

[0116] In non-limiting embodiments, the drug is an antiviral agent (which as used herein includes antiretroviral agents), such as adamantane antivirals, antiviral boosters, antiviral combinations, antiviral interferons, chemokine receptor antagonists, integrase strand transfer inhibitors, miscellaneous antivirals, neuraminidase inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), non- structural protein 5A (NS5A) inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors, and / or purine nucleosides. Exemplary antiviral agents are known to those of skill in the art.

[0117] In non-limiting embodiments, the drug is an anti-inflammatory agent, such as, without limitation, an NSAID, such as salicylic acid, indomethacin, sodium indomethacin trihydrate, salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, sodium salicylamide; an anti-inflammatory cytokine; an anti-inflammatory protein; a steroidal anti-inflammatory agent; or an anti-clotting agent, such as heparin. Other drugs that may promote wound healing and / or tissue regeneration may also be included.

[0118] In non-limiting embodiments, one or more cells are added to the vECM-containing composition. As used herein, the terms “cell” and “cells” refer to any types of cells from any animal, such as, without limitation, pig, rat, mice, monkey, and human. For example and without limitation, cells can be progenitor cells, such as stem cells, or differentiated cells, such as endothelial cells and smooth muscle cells. In non-limiting embodiments, cells for medical procedures can be obtained from the patient for autologous procedures or from other donors for allogeneic procedures.

[0119] Non-limiting examples of useful cells include fibroblasts, epithelial cells, stem cells, multi-potent cells, progenitor cells and differentiated cells; recombinant (e.g., genetically engineered) cells; muscle cells and precursors thereof; nerve cells and precursors thereof; mesenchymal progenitor or stem cells; pre- adipocytes, and the like. In non-limiting embodiments, the cell is one or more cells naturally found in vaginal tissue (e.g., basal cells, parabasal cells, superficial squamous flat cells, and / or intermediate cells).

[0120] Any useful therapeutic agent can be mixed into, mixed with, co-applied, or otherwise combined with any composition as described herein. For example, and without limitation, useful components include vECM and one or more growth factors, interferons, interleukins, chemokines, monokines, hormones, angiogenic factors, and / or drugs. Cells can be mixed into the composition or can be included on or within a substrate such as a biological scaffold, combined with the composition. In any case, when the substrate is seeded with cells, the cells can be grown and / or adapted to the niche created by incubation in a suitable medium in a bioreactor or incubator for a suitable period to optimally / favorably prepare the composition for implantation in a patient. The substrate can be seeded with cells to facilitate in-growth, differentiation and / or adaptation of the cells. The cells may be autologous or allogeneic with respect to the patient to receive the composition / device comprising the vECM-containing composition. The cells may be stem cells, pluripotent cells, multipotent cells, progenitor cells, and / or differentiated cells.

[0121] In a non-limiting embodiment, a commercial kit is provided comprising a composition described herein. A kit comprises suitable packaging material and the composition. In non-limiting embodiments, the kit comprises a liquid, gelled or dried vECM in a vessel, which may be the packaging, or which may be contained within packaging. The vessel may be a vial, syringe, tube or any other container suitable for storage and transfer in commercial distribution routes of the kit. Likewise, a product, such as a device, gel, scaffolding, suture, prosthetic, mesh, foam etc. including the vECM-containing compositions described herein may be packaged appropriately for commercial distribution.Example

[0122] Results of decellularization (H&E staining) by the foregoing methods are shown in FIG. 2, where NT = no decellularization, FT = freeze-thaw, SDC = detergent treatment, and Hybrid = freeze-thaw and detergent. In addition to the H&E staining shown in FIG. 2, reduction of double stranded DNA (dsDNA) by each method is shown below in Table 1.Table 1

[0123] In addition to the foregoing, the various treatments for removing cellular components were evaluated for collagen content, as shown in FIGS. 3A-3B and glycosaminoglycan (GAG) content, as shown in FIGS. 4A-4B. As can be appreciated by the results, collagen type I becomes a greater portion of vECM mass following decellularization, while stratification by vaginal region did not identify any region- specific differences. With regard to GAG content, hybrid decellularization provides the most robust reduction in GAG content.

[0124] As can be appreciated from the foregoing results, physical decellularization (freezethaw) provides the least efficient dsDNA, though the resultant materials include high levels of collagen and GAG, thus retaining gross mechanical properties. Hybrid decellularization provides a more efficient reduction in dsDNA, though higher levels of GAG loss are seen, which alters gross mechanical properties. Lastly, the detergent-based method provides highly efficient dsDNA reduction, with high levels of collagen and GAG, thus retaining gross mechanical properties.

[0125] In non-limiting embodiments, as described herein, the pre-gel solution may be freeze dried again. Once gelation is desired, such gelation is induced by bringing the hydrated gel to neutral pH and physiologic salinity, where the pre-gel may gel in minutes. This method of fabricating a gel has near unlimited customizability, e.g. through addition of donor cells, growth factors and pharmaceuticals, or as a coating for exogenous surgical repair materials (e.g., enhancing vaginal integration of biomaterial augmented urogynecologic procedures). The mechanical properties of the hydrogel can also easily be adjusted, e.g. through alteration of vECM concentration within the pre-gel, addition of crosslinking compounds, or altering thegel salinity. These hydrogels can be cast in multiple sizes, ranging from small constructs appropriate for cell culture or sheets with high surface area used to coat other surgical repair materials. We believe that further development of this technology will be highly useful as a research tool for developing surgical devices to repair POP or alternatively as a medical device to regenerate injured or impaired vagina.

[0126] Without wishing to be bound by the theory, the decision to utilize vaginal tissue rather than a more common protein source for organ-derived hydrogels (i.e. bladder, intestine) provides a number of advantages, as reproducing the unique vECM composition is conducive to promoting proper wound healing over time and measuring accurate cell response in vitro. Hydrogels may be fabricated at varying concentrations that correspond to varying stiffnesses, including at concentrations ranging from about 5 mg / mL to about 25 mg / mL, from about 10 mg / mL to about 25 mg / mL, from about 10 mg / mL to about 20 mg / mL, from about 10 mg / mL to about 15 mg / mL, from about 15 mg / mL to about 20 mg / mL, all values and subranges therebetween included, corresponding with increasing stiffness as measured via rheometer. Without wishing to be bound by the theory, as discussed herein the use of a second lyophilization step (e.g., lyophilizing the digested, decellularized ECM), allows for inclusion of ECM at a higher concentration that is otherwise possible, while still providing a workable material that exhibits both acceptable mechanical properties (e.g., rheology and / or adequate delamination thresholds) and is conducive for use as a cell scaffold. In non-limiting embodiments, the scaffolds as described herein do not include vECM at less than 5 mg / mL. In non-limiting embodiments, gels, with or without embedded cells, may be tunable to be specific to a particular use and / or condition to be treated. This may be seen in FIGS. 5-6.

[0127] In non-limiting embodiments, a gel formed of vECM at concentrations of 10 mg / mL, 15 mg / mL, and 20 mg / mL exhibit a storage modulus (measured using a rheometer) of 170 Pa, 117 Pa, and 57 Pa, respectively, when a second lyophilization step is not included in the decellularization / solubilization process. In non-limiting embodiments, when a second lyophilization step is included (e.g., following solubilization with an acid protease), vECM gels at a concentration of 5 mg / mL and 10 mg / mL exhibit a storage modulus of 129.556 ± 2.8 Pa and 218.28 ± 25.3 Pa, respectively, as shown in FIGS. 7-8. As also shown in FIGS. 7-8, the addition of a second lyophilization step may provide a longer working time (shown in the blue line), which may allow for a healthcare professional to work with the ECM material for a greater amount of time before gelation occurs.

[0128] As discussed above, hydrogels formed from vECM may be seeded with cells, for example fibroblasts. As shown in FIG. 9, hydrogels formed from vECM seeded withfibroblasts exhibit good cell penetration. In FIG. 9, the left panel shows a gel formed of 15 mg / ml vECM, while the right panel shows a gel formed of 20 mg / mL vECM. As shown in FIGS. 10-15, a hydrogels (5 and 10 mg / mL vECM) demonstrated ability to proliferate and interact with surrounding matrix in 5 and 10 mg / mL gels (FIG. 10), with viability confirmed by apoptosis marker (Caspase-3) indicating less than 14% cell death by day 14 (FIG. 11), as well as activation and proliferation of cells, on days 1, 7, and 10 as indicated by myofibroblast markers (aSMA and vimentin) FIG. 12.

[0129] As discussed above, hydrogels formed from vECM may be applied to prosthetic materials, such as meshes. Shown in FIG. 15 is a polypropylene mesh coated in vECM.Example 2

[0130] vECM Decellularization: Vaginas were harvested from gilt pelvic viscera acquired from a local abattoir (Thoma Meat Market, Saxonburg, PA, USA). Each organ was minced into 1 mm3 pieces and all tissue was pooled prior to decellularization. Tissue was incubated in sterile ddH2O at 4C for 48 hours, with the samples shaken and ddfUO replaced every 24 hours. A modified version of Meder et al.’s procedure was then followed, which consisted of successive immersions in 0.02% Trypsin (Thermo Fisher Scientific, Waltham, MA) in phosphate-buffered saline (PBS) at 37C, 3% Triton-X-100 (Thermo Fisher) in sterile ddthO, 4% sodium deoxycholate (Thermo Fisher) in sterile ddthO, and 90 / 10 / 0.26% sterile ddthO / ethanol / peracetic acid, (Thermo Fisher) with alternating washes in ddthO and sterile PBS after each step. Decellularization was considered finished when the tissue turned pure white and agitating the tissue in ddthO did not create any residual bubbles. The resulting vECM was lyophilized for 48h in a FreeZone Freeze Dryer (LabConco, Kansas City, MO) and milled to a powder via Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ) with a metal screen of 20 squares / inch.

[0131] vECM Gelation: Powdered vECM was digested in a solution of 0.01N HC1 1000 U / mL Pepsin for 48 hours under constant stirring before aliquoting and lyophilizing. Lyophilized pre-gel pellets were weighed and reconstituted to desired concentration (5 and 10 mg / mL) using a solution of half UltraPure Distilled Water and half supplemented media for cell viability during the gelation process. Because of the fraction of media, the volume of media was modified to accommodate for a final 1 / 10 salt concentration. Gels were then neutralized with 0.1N NaOH using an empirically found NaOH value that did not exceed 1 / 9 volume. The gels are then placed in 13mm diameter stainless steel molds and incubated at 37°C for 30 min- 1 hour to allow gelation. These gels served as acellular controls and were removed from study at days 1, 7, and 14 and preserved for histology, biochemistry, or RNA as indicated below.

[0132] Biochemical analysis of vECM components: 5 mg lyophilized portions of tissue and vECM, and whole gels were digested in papain enzyme solution at 65°C overnight. Papain digestions were performed at 10 mg sample per 1 mL solution. After digestion, samples were cooled and centrifuged to pellet undigested solids. The liquid digest was used for colorimetric biochemical analysis and the remaining tube and pellet weight were used to determine the mass of gel actually digested. 1,9-dimethyl methylene-blue (DMMB) precipitation assay was performed on papain digested samples to determine sulfated glycosaminoglycan (sGAG) concentration by percent weight. Samples were run in duplicate alongside a chondroitin sulfate reference standard (Biocolor, Carrickfergus, UK) until readouts were within 10%. Percent sGAG weight of all samples was determined after normalization to mass of sample digested. For acellular gels, additional normalization was performed to account for contributions of non- vECM constituents (e.g. pepsin, salt, media components). Hydroxyproline assay was performed on papain digested samples to determine collagen I concentration by percent weight. HC1 digest as described previously, using perchloric acid was used. Samples were run in duplicate alongside rat tail collagen and hydroxyproline (Sigma-Aldrich) standards until readouts were within 10%. Normalizations were performed per sGAG quantification.

[0133] dsDNA quantification: 5 mg lyophilized portions of tissue and vECM, and whole gels were processed via QIAGEN DNEasy Extraction Kit (QIAGEN, Hilden, Germany) per manufacturer’s instructions. Each sample was eluted 5 times. Sample elutions were run in duplicate via Quant-iT PicoGreen dsDNA Assay (Thermo Fisher) alongside provided calf thymus DNA standard until readouts were within 10%. dsDNA content of each elution was reported in ng / mg dry sample weight, and five elutions were summed to provide the final dsDNA content of each sample. Two successive dsDNA elutes from tissue, vECM, and 5 mg / mL and 10 mg / mL acellular vECM hydrogels were diluted in 6x loading dye based on PicoGreen assay output and run alongside a Quick-Load® Purple 100 bp DNA Ladder diluted in loading dye and DNAse free water. All samples were run on a 3% low melting point agarose gel with ethidium bromide at 60 V for three hours and visualized via UV transillumination.

[0134] Statistical analysis entailed one-way ANOVA followed by Bonferroni post-hoc.

[0135] Results are shown in FIGS. 16A-16C. Vaginal biopsies from nulliparous porcine juveniles were excised and processed for decellularization. FIG. 16A (Panels A and E) illustrates gross morphology of the intact vagina and the vagina post-decellularization, respectively, showing that rugae definition and gross mechanical properties were qualitatively preserved after vECM decellularization. FIG. 16A (Panels B and F) shows histology samples stained with H and E to label nuclei (purple) and cytoplasmic and matrix proteins pink. FIG.16A (Panel B) evidences absence of cellular contents in vagina, and Panel F shows a massive decrease in nuclei after decellularization. FIG. 16A (Panels C and H) shows use of picrosirius red to visualize collagen (red) showing maintenance of collagen before (Panel C) and after (Panel H) decellularization. Picrosirius red staining visualized with polarized light (FIG. 16A, Panels D and H) shows collagen maturation, with mature collagen appearing red and less mature or newly deposited collagen appearing green. Picrosirius red stained vECM appeared subjectively redder under circularly polarized light, indicating some partial distortion of the ECM micro structure during the decellularization process resulting in thickened collagen fibers. Brightfield and polarized images were acquired at 10X. All scale bars 500 pm. FIG. 16B (Panel I) shows collagen I content determined biochemically maintained through the decellularization process by dry weight. Collagen I content was significantly higher in vECM (87.3 ± 5.7%; p=0.008) and hydrogels (84.1 ± 12.4%; p=0.03) compared to tissue (69.6 ± 6.1%), with no differences between vECM and hydrogel. FIG. 16B (Panel J) shows no significant differences in sGAG content were noted (tissue 1.3 ± 0.3%; vECM 1.3 ± 0.2%; gels 1.2 ± 0.3%). FIG. 16B (Panel K) shows Picogreen assay to determine dsDNA quantity showed reduced DNA content by 75.8% in vECM and 100% in gel (p<0.001). This figure thus shows successful decellularization and confirms gels contained effectively zero dsDNA. Control tissue samples were confirmed to contain genomic data larger than the maximum ladder size. In the first elute of decellularized vECM, the majority of remaining DNA fragments were below 200 bp, within the acceptable range for dsDNA content post-decellularization. The first elute of the 5 mg / mL and 10 mg / mL gels did not contain any visible bands indicating no dsDNA (FIG. 16C).

[0136] Mass spectrometry evidence of tissue, vECM, and vECM gel composition. Tissue. vECM, and 10 mg / mL vECM hydrogels rehydrated without media were lyophilized for 48h and submitted to the University of Pittsburgh Health Sciences Mass Spectrometry Core for tandem mass tagging and label-free quantitation of abundant proteins. Peptide fragment sequences were identified and used to determine proteins of origin. Results are displayed below in Table 2.Table 2All identified proteins were from porcine sources. 92 unique proteins were detected in gel,2653 in tissue, and 2425 in vECM. The 14 most abundant proteins in the hydrogel sample, ordered by spectral count, are displayed. UniProt ID mapping was performed to acquire Gene Ontology (cellular component) annotations for each protein. As expected, extracellular matrix proteins had higher spectral count in the gel sample compared to tissue. Decellularized vECM also demonstrated higher spectral count of matrix proteins than tissue but lower than gel, andcytoplasmic proteins including myosin 11 and cytoplasmic actin had nonzero but low spectral counts in the gel. We attribute these observations to our decellularization efficiency being less than 100% resulting in scant retained intracellular protein fragments. We attribute the high spectral count of proteins including pepsin A, trypsin, carbamoyl-phosphate synthase, and chitinase present in the gel to the enzymatic steps required for decellularization and hydrogel synthesis. Growth factors including TGFB and FGF were not detected in gel indicating that this material is a blank slate for pharmacological modification.

[0137] Rheological assessment: Gelation kinetics were found using a TA AR2000 Dynamic Rheometer as described.40 Briefly, 1 mL of vECM hydrogel was placed into the chamber kept at 17 °C. The parallel plate (40 mm) was brought into contact with the sample at 500pm. The shear storage (G’) and loss moduli (G”), which characterize gel elastoplastic properties, were measured while the plate oscillates at 1 Hz and 5% strain. A 10 min temperature hold was performed to demonstrate sufficient pre-gelation working time, the temperature then increased at 2°C / min until 37°C to assess gelation temperature and kinetics for 10 minutes, then held at 37C for 30 minutes. The max shear storage during the 30-minute hold at 37°C was used to determine the stiffness value.

[0138] Protein Quantification: Bulk protein quantification was performed by briefly homogenizing samples in a 0.15M NaCl and 50mM Tris Base extraction buffer containing protease inhibitors. Samples were then run through a DC protein assay (BioRad, Hercules, CA) to quantify protein, and therefore concentration of protein per constructed vECM gel.

[0139] Results are shown in FIGS. 187-18C. FIG. 17A shows the overlay of gelation curves shows the storage modulus (blue), as determined by rheological analysis, measured the elastic properties of vECM hydrogels fabricated at concentrations ranging from 2 to 14 mg / mL. This storage modulus was found after a 10-minute temperature hold at 17, a 10-minute, 2- degree / minute temperature ramp, and a 30-minute hold at 37°C under 1 Hz oscillation and 5% strain. This demonstrates sufficient pre-gelation working time prior to gelation. FIG. 17B shows analysis of the data shows a logarithmic relationship between protein concentration and storage modulus. This data demonstrates a correlation between vECM concentration and gel stiffness, thereby providing a means to control the mechanical properties of the hydrogels for specific applications or individual requirements. FIG. 17C shows detergent-based protein assay supports that vECM gels can be successfully fabricated at varying concentrations. Specifically, gels prepared at 5 mg / mL and 10 mg / mL are biochemically found to be at a vECM concentration of 4.52 ± 0.5 mg / mL and 8.9 ± 0.9 mg / mL, respectively.

[0140] vECM Hydrogel processing and SEM procedure: Hydrogels were fixed in 2.5% glutaraldehyde followed by dehydrating ethanol washes and imaged via JEOL JSM 6335F scanning electron microscope (SEM) (accelerating voltage 3 keV) at IkX, 7.5kX, and 20kX after critical point drying and 5 nm Pd-Au sputter coating. A custom MATLAB R2023a code was used to quantify gel fiber diameter and porosity of three ROIs per gel.

[0141] Statistical analysis entailed Student’s t-test.

[0142] Results are shown in FIGS. 18A-18B. Acellular 5mg / mL and 10 mg / mL vECM hydrogels were fixed in 2.5% glutaraldehyde and subjected to graded ethanol wash prior to critical point drying and 5 nm gold-palladium sputter coating for scanning electron microscopy (SEM). 3 ROIs were acquired for each gel (N=3 / concentration). FIG. 18A shows of acellular 5mg / mL and 10 mg / mL vECM hydrogels at 1000X (top), 7500X (middle), and 20,000X (bottom). As expected, hydrogel micro structure had a uniform density of microfibers with no clear orientation or crystallinity, supporting the claim that this device can be fabricated in a replicable manner. SEM image analysis was conducted via custom MATLAB 2023a code to assess fiber porosity, average fiber diameter (AFD), and connectivity. FIG. 18B shows, microscopically, 5mg / mL gels had significantly higher porosity (p=0.0210) and AFD (pcO.0001) than lOmg / mL gels. 5mg / mL gels had significantly lower fiber connectivity (pcO.0001). The inverse relationship between porosity and fiber connectivity was expected.

[0143] Human Vaginal Fibroblasts (HVFs): A full-thickness vaginal punch biopsy was taken from human patients as part of the Magee-Women’s Research Institute Pelvic Floor Biorepository (IRB: CR19080175-010). The epithelium is removed before mincing the tissue section in supplemented media (Dulbecco’s Modified Eagle Medium (DMEM), 14% Fetal Bovine Serum (FBS), 1.5% Amphotericin B, 1% Pen-Strep). Fibroblasts are determined as outgrowths from tissue sections after 10 days, isolated using trypsin, and replated. Cells lines used for this study (N=3) were from pre-menopausal patients with no topical estrogen use and no diagnosis of pelvic organ prolapse. Cells were between passages 4 and 8 to avoid senescence, with the entire study being done with the same passage for each line.

[0144] 3D seeding of HVFs: Cells were grown out to 80% confluence before being passaged using trypsin and counted. Cells were resuspended in supplemented DMEM to allow seeding 250,000 cells per gel. Cells were resuspended in the neutralized gels, placed in 13mm stainless steel molds and incubated at 37°C for 30 min-1 hour. These gels served as cellular gels and were removed from study at days 1, 7, and 14 and preserved for histology, biochemistry, or RNA as indicated below.

[0145] SEM imaging of acellular and cellular vECM hydrogels: Acellular 5mg / mL and lOmg / mL gels and cellular 5mg / mL and lOmg / mL gels seeded with human vaginal fibroblasts at 250,000 cells / gel were cultured for 14 days. Before scanning electron microscopy, all samples were fixed in 2.5% glutaraldehyde and washed with IX PBS. Cellular gels underwent additional fixation in 1% osmium tetroxide. Samples were then washed with IX PBS followed by a dehydrating graded ethanol wash. After critical point drying, samples were sputter-coated with a 5nm gold-palladium alloy. Each sample was imaged via JEOL JSM 6335F at 3.0 kV across three random regions at l,000x, 7,500x, and 20,000x. Image analysis was conducted via custom MATLAB 2023a code to assess fiber porosity, average fiber diameter, and connectivity. vECM hydrogels were imaged via brightfield microscope to measure contraction of both cellular and acellular 5 mg / mL and 10 mg / mL gels. Image analysis was conducted using QuPath 0.5.1 with an ImageJ plugin, with outputs of Feret’s diameter, area, and perimeter.

[0146] Calculation of Hydrogel Contraction: A brightfield dissection microscope (Bioimager Inc, Ontario, Canada) was used to image acellular and cellular hydrogels at 5 mg / mL or 10 mg / mL concentration every day for 14 days. Image analysis was performed via QuPath 0.5.1 with an ImageJ plugin to measure Feret’s diameter, area, and perimeter. Percent reduction for diameter, area, and perimeter were calculated by normalizing to Day 0 measurements. Statistical analyses were conducted using Prism 10, employing one-way repeated measures ANOVA followed by Tukey's post-hoc test for comparisons.

[0147] Statistical analysis entailed one-way ANOVA followed by Bonferroni post-hoc.

[0148] Results are shown in FIGS. 19A-19B. Acellular 5mg / mL and lOmg / mL gels and cellular 5mg / mL and lOmg / mL gels seeded with human vaginal fibroblasts at 250,000 cells / gel were cultured for 14 days. Acellular hydrogels did not contract a statistically significant amount over the 14-day period. All cellular hydrogels contracted exponentially over the same period (average R2=0.8962). Statistically significant decreases in all outcomes (area, diameter, perimeter) were observed for 5 mg / mL and 10 mg / mL conditions at day 7 and 14 timepoints, and 5 mg / mL cellular gels had lower area, perimeter, and diameter than 10 mg / mL cellular gels at all timepoints (FIG. 19A). Scanning electron micrographs show a distinct change in cellular lOmg / mL gel micro structure at day 14 of culture, and brightfield imaging parallels the contraction data (FIG. 19B). These results indicate that gel morphology is capable of modification by introduced cells and they are greatly promising for this technology as tunable research platform or clinical device.

[0149] Histology preparation and staining: Gels were fixed for 24 hours in 10% Formalin and stored in 100% ethanol until being paraffin embedded on two orientations: cross-section to look at the entire surface area and longitudinally to capture the dispersion throughout the entirety of the gel and sectioned at 7 pm. Samples were de-paraffinized at 20-22°C for 20 minutes and rehydrated. Sections were H&E stained for four minutes in Gill’s hematoxylin and two minutes in eosin and imaged via Motic Easy Scan Pro Digital slide scanner (Feasterville, PA, USA) at 40x. Cell density was quantified using open-source image analysis software QuPath (QuPath v0.4.3).l Additional sections were stained for one hour in picrosirius red and imaged via circularly polarized light on a Nikon Eclipse NiE upright microscope (put manufacturer name and location) at 10X (exposure 40 ms, lamps 40). Differential collagen deposition of thick versus thin fibers in vaginal adventitia was evaluated by calculating the amount of red vs green pixels using MATLAB code previously developed in a prior study.2

[0150] Immunofluorescence: Paraffin embedded tissue sections were labeled with vimentin and a-smooth muscle actin (aSMA). Embedded tissue was deparaffinized and antigen retrieval was performed with citric acid antigen buffer (at 95-100°C for 20 minutes). Buffer was cooled to 37°C and washed with TBST (IX Tris buffered saline / 0.1% Tween 20, pH 7.4). Tissue was blocked with 2% Bovine Serum Albumin (BSA) and 0.1% Tween for 2 hours at room temperature and incubated with vimentin (MA5-11883, Invitrogen, Waltham, MA) and alpha smooth muscle actin (ab21027, Abeam, Cambridge, UK) overnight at 4 °C. After overnight incubation, slides were washed and incubated in secondary () at room temperature for 90 minutes. Sections were washed and mounted using aqueous mounting medium with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA) before cover slipping. The entire gel was imaged using Nikon Eclipse NiE Upright Microscope (Nikon USA, Melville, NY) at 20X. Number of vimentin+ and / or aSMA+ per total cells (DAPI only) were quantified by thresholding out non-specific labelling and background using QuPath (QuPath v0.4.3).

[0151] Results are shown in FIGS. 20A-20C. Human vaginal fibroblasts seeded at 250,000 cells / gel in 5-25 mg / mL gels demonstrated greater ability to proliferate and interact with surrounding matrix in 5 and 10 mg / mL gels compared to 20-25 mg / mL gels (FIG. 20A). At day 0, no colocalization of myofibroblast or apoptosis markers was observed (FIGS. 20B- 20C). By day 7, cells attached to the matrix, expressing myofibroblast markers in vECM hydrogels (vimentin plus aSMA, indicating differentiation and activation of the cells in vECM hydrogels, with acceptable yield of cell death (<14%) by day 14 (FIG. 20B). Time-course images of lOmg / mL and 20mg / mL gels over 14 days showed enhanced cell contraction inlOmg / mL gels (FIG. 20C). The results indicate the successful fabrication of a tunable in vitro system to test vaginal fibroblast behavior by modifying the concentration of protein to modulate mechanical properties using a relevant homologous tissue source. Further work seeks to ascertain more information about the activation state through cellular outputs and the regulatory processes activated by mechanical cues to further develop in vitro modeling devices. Use of hormones and other factors (growth factors, matrix bound vesicles, etc) is ongoing.

[0152] Injection of vECM hydrogel: Porcine vECM hydrogel was injected into the posterior vaginal subepithelium in two C57BL / 6 mice FIG 21 A. Tissue was harvested at 2 days and compared to two control mice without intervention. H&E staining on two sections per mouse demonstrated an expected infiltrate at the site of injection with no adverse events.

[0153] Fluorescein imaging: After subepithelial vaginal injection of 0.2% fluorescein + porcine vECM hydrogel into four C57BL / 6 mice, IVIS fluorescent imaging was performed at 1-day post- injection demonstrating hydrogel presence in the vagina but not in the spleen, liver, kidneys, or uterus FIG. 21A.

[0154] Results are shown in FIGS. 21A-21B. Florescein tagged porcine vECM hydrogel was injected into the posterior vaginal subepithelium in two C57BL / 6 mice. After subepithelial vaginal injection of 0.2% fluorescein + porcine vECM hydrogel into four C57BL / 6 mice, IVIS fluorescent imaging was performed at 1-day post-injection demonstrating hydrogel presence in the vagina but not in the spleen, liver, kidneys, or uterus. This evidences hydrogel localization to the site of injection at 24 hours with no off-target presence in the whole body (FIG. 21A, Panel A) or dissected organs (FIG. 21A, Panel B). Tissue was harvested at 2 days and compared to two control mice without intervention (FIG. 21B). H&E and trichrome staining on two sections per mouse demonstrated an expected infiltrate at the site of injection with no adverse events.

[0155] Restorelle polypropylene mesh and a prototype biocompatible elastomeric polycarbonate urethane (PCU) membrane were embedded in acellular 5 mg / mL and 10 mg / mL vECM hydrogels. After gelation occurred, the gel-mesh constructs were loaded into a 3D printed uniaxial tension device and 20% static tension was applied. Tension was then removed and the constructs were macroscopically analyzed for damage to the mesh or gel. SEM image analysis of gel-mesh constructs and unfunctionalized hydrogels was conducted via custom MATLAB 2023a code to assess fiber porosity, average fiber diameter, and connectivity. Additional Restorelle® mesh was embedded in acellular 10 mg / mL hydrogel and the coating was air dried after gelation to form a thin transparent film. The dry gel-mesh construct was placed in saline and immediately rehydrated prior to rough manipulation by a urogynecologicsurgical fellow, which involved insertion through a 5 mm laparoscopic port, twisting, and stretching using laparoscopic forceps. Additional PCU elastomeric membranes were coated with gel, dried, and immersed in saline to assess coating degradation at 37 °C in saline.

[0156] Results are shown in FIG. 22. Restorelle® polypropylene mesh and a prototype biocompatible elastomeric polycarbonate urethane (PCU) membrane were embedded in acellular 5 mg / mL and 10 mg / mL hydrogels. After gelation occurred, the gel-mesh constructs were loaded into a 3D printed uniaxial tension device and 20% static tension was applied. No gross delamination of the gels occurred, and the gel-mesh constructs displayed pore collapse and wrinkling consistent with deformation of uncoated mesh and membranes (FIG. 22, Panel A). After tension was removed, the gel-mesh constructs successfully returned to their undeformed configurations. SEM image analysis of gel-mesh constructs and unfunctionalized hydrogels was conducted via custom MATLAB 2023a code to assess fiber porosity, average fiber diameter (AFD), and connectivity. No significant difference in porosity, average fiber diameter, or fiber connectivity were observed (FIG. 22, Panel B). Additional Restorelle® mesh was embedded in acellular 10 mg / mL hydrogel and the coating was air dried after gelation to form a thin transparent film (FIG. 22, Panel C). The dry gel-mesh construct was placed in saline and immediately rehydrated. The rehydrated construct maintained its form after simulated rough manipulation by a urogynecologic surgical fellow, which involved insertion through a 5 mm laparoscopic port, twisting, and stretching using laparoscopic forceps (FIG. 22, Panel D). PCU elastomeric membranes were coated with gel and dried using the same method. The coated membranes also held up to rough handling, and the gel coating remained intact for 4 (FIG. 22, Panel E), 8 (FIG. 22, Panel F), and 10 (not shown) days at 37°C rocking in saline. Next steps include optimizing the uniaxially tensioned gel-mesh constructs as platforms for investigating cell response to mesh and prototype mesh alternatives such as PCU membranes. Further work to characterize the microstructure, surface mechanics, and animal host response to rehydrated gel-coated mesh and PCU membranes is ongoing.

[0157] Primary adult human vaginal fibroblasts (HVFs) derived from a premenopausal patient without diagnosis of pelvic organ prolapse were used with permission from the Magee- Women’s Research Institute Pelvic Floor Biorepository. Primary neonatal human foreskin fibroblasts (HFFs) were purchased from Gibco / Thermo Fisher Scientific. 50,000 cells were seeded directly on 14mm diameter hydrogel pucks and allowed to adhere in serum- supplemented media (phenol red-free Dulbecco’s Modified Eagle Medium (prfDMEM), 14% fetal bovine serum (FBS), 1.5% Amphotericin B, 1% penicillin / streptomycin). Media was then changed to prfDMEM with 1.5% Amphotericin B and 1% penicillin / streptomycinsupplemented with either 14% FBS or 0.2% w / v lactalbumin enzymatic hydrolysate (LAH) and either 0, 200, or 400 pg / mL 17-P-estradiol (E2) (N=4 per line per media condition). LAH is derived from hydrolyzed whey protein and was chosen as a control media supplement due to its lack of hormones and cytokines. Gel diameter was measured at day 4 of culture as an indication of fibroblast activity. HFFs and HVFs were cultured for four days on standard tissue culture plates for 4 days in media with either FBS or LAH and either 0, 200, or 400 pg / mL E2 (N=3 per line per media condition). Cell viability was assessed via ethidium homodimer assay followed by comparison of fluorescent signal ratios in ImageJ. Additional representative cell- seeded gels were cultured in prfDMEM with FBS supplementation and embedded in Tissue- Tek O.C.T. Compound after 1 or 7 days in culture. 20 pm cryosections were stained with H&E and PSR and 7 pm cryosections were labeled with DAPI. All slides were imaged at 10X. Statistical analysis entailed one-way ANOVA followed by Bonferroni post-hoc or t-test.

[0158] Results are shown in FIGS. 23A-23C. Primary human foreskin (HFF) or vaginal fibroblasts (HVF) were seeded on top of gels in phenol red free DMEM containing 14% FBS and allowed to adhere. On day 1 of culture, 0, 200, or 400pg / mL 17-P-estradiol (E2) containing media was then added in the presence (FBS) and absence (LAH) of serum for 4 days with gel diameter measured at day 4. Growth of HFFs and HVFs cultured under identical media conditions without hydrogels were compared and cell viability was assessed via ethidium homodimer assay. Representative 14 mm gels seeded with HFFs or HVFs were cultured in DMEM with FBS for 1 or 7 days and stained via H&E, DAPI, and picrosirius red. H&E stained HVF and HFF-seeded gels embedded at day 1 of culture showed a gel microstructure similar to that of acellular gels, with cells only apparent on the gel surface. HVF and HFF-seeded gels embedded at day 7 of culture had greater cell density at their surface and cells had also infiltrated deeper into the gel microstructure. DAPI labeling verified this observation (Figure 8A-H; arrows on DAPI images indicate nuclei). Polarized light imaging of picrosirius red stained gels showed faint, mixed green and red fibers distributed evenly throughout the gel volume at day 1. vECM hydrogel fibers appeared thickened and darker at the site of seeding at day 7 and redder under polarized light (Panels LP). All images are at 10X; scale bar 200 pm. FIGS. 23B-23C show increased E2 concentration in media resulted in greater gel contraction regardless of FBS or LAH supplementation. At day 4 of culture, gels had contracted by an average of 33.9 ± 10.9%, with HVF-seeded gels cultured in prfDMEM / FBS supplemented with 0 pg / mL or 400 pg / mL E2 contracting the greatest. In prfDMEM supplemented with either FBS or LAH, HVF-seeded gels underwent significantly greater percent contraction than HFF- seeded gels when either 0 pg / mL (p=0.001 (FBS); p=0.00009 (LAH)) or 200 pg / mL E2 (p=0.04(FBS)) was added. However, addition of 400 pg / mL E2 to cell media eliminated these significant differences regardless of FBS or LAH. Within the HFF group, increasing E2 resulted in statistically significant increases in contraction regardless of serum for both 0 to 200 pg / mL (FBS p=0.0006; EAH pcO.OOOl) and 0 to 400 pg / mL (FBS pcO.OOOl; EAH pcO.OOOl) (FIG. 23C). Within the HVF group in LAH supplemented media (FIG. 23C), increasing E2 concentration from 0 to 200 pg / mL resulted in significantly increased gel contraction (p=O.O183), an effect which was not seen with further increase to 400 pg / mL or for FBS supplementation. Serum supplementation resulted in increased percent contraction for either cell type controlling for E2 supplementation. No significant differences in cell viability were observed regardless of cell line or media condition.

[0159] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS1. A method of treating a condition in a patient, comprising administering to the patient, an extracellular matrix (ECM) composition comprising decellularized, acid- protease-digested ECM derived from vaginal tissue.

2. The method of claim 1, wherein the vaginal tissue is human, bovine, ovine, or porcine.

3. The method of claim 1, wherein the acid-protease-digested ECM is not dialyzed or chemically crosslinked.

4. The method of claim 1, wherein the composition forms a gel when warmed to 37°C.

5. The method of claim 1, wherein the ECM composition is applied to a mesh or graft that is implanted in the patient.

6. The method of claim 5, wherein the mesh or graft comprises an inorganic material.

7. The method of claim 5 or claim 6, wherein the ECM composition is coated onto a surface of the mesh or graft.

8. The method of claim 1, wherein the ECM composition is seeded with one or more cells.

9. The method of claim 8, wherein the one or more cells comprise one or more fibroblasts.

10. The method of claim 1, wherein the ECM composition comprises one or more therapeutic agents.

11. An extracellular matrix (ECM) gel comprising acid-protease-digested ECM derived from vaginal tissue and one or more cells dispersed therein.

12. A method of preparing an extracellular matrix (ECM) material, comprising: a. incubating vaginal tissue in a protease;b. incubating the vaginal tissue in a solution comprising at least one detergent; c. incubating the vaginal tissue in a composition comprising at least one disinfecting agent, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e. comminuting the lyophilized ECM material; f. partially or completely solubilizing the comminuted, lyophilized ECM material with an acid protease to produce solubilized ECM; and g. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

13. A method of preparing an extracellular matrix (ECM) material, comprising: a. exposing vaginal tissue to at least one freeze-thaw cycle; b. disinfecting the vaginal tissue, optionally with at least one antibiotic and / or antimycotic composition; c. incubating the vaginal tissue in a solution comprising at least one detergent, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e comminuting the lyophilized ECM material; f. partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM; and g. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

14. The method of claim 13, further comprising, after lyophilizing the solubilized ECM, neutralizing the lyophilized solubilized ECM to produce an ECM pre-gel.

15. The method of claim 14, further comprising gelling the ECM pre-gel at a temperature at which the ECM pre-gel gels to produce an ECM gel16. The method of claim 13, wherein: the detergent is a zwiterionic detergent and / or an anionic detergent, optionallyCHAPs, SDS, sodium deoxycholate, and / or Triton-X-100; and / or the disinfecting agent is ethanol and / or peracetic acid.

17. The method of claim 13, wherein the decellularized ECM material is not completely digested with the acid protease, producing an ECM pre-gel that is able to gel at 37°C comprising undigested decellularized ECM particles.

18. The method of claim 13, further comprising including one or more washing steps from prior to the step of comminuting the lyophilized ECM material.

19. The method of claim 18, wherein the one or more washing steps comprises washing the tissue or material with phosphate-buffered saline, saline, and / or water.

20. The method of claim 13, wherein the ECM material is prepared without a dialysis step or a crosslinking step.

21. The method of claim 13, wherein the acid protease is pepsin and / or trypsin.

22. The method of claim 13, wherein the lyophilized ECM material is solubilized with an acid protease in a solution having a pH of from 1 to 4, from 1 to 2, or 2.0 ± 0.3.

23. The method of claim 13, comprising dispersing the ECM material in a natural or a synthetic polymer composition.

24. The method of claim 23, wherein the natural or a synthetic polymer composition is one or more of: a second ECM material, fibrin, collagen, polyester (PE), polyurethane (PU), poly(ester urethane) urea (PEUU), poly(ether ester urethane) urea (PEEUU), poly (ester carbonate urethane)urea (PECUU), poly (carbonate urethane)urea (PCUU) copolymer, polyolefin (poly alkene), polycarbonate, poly anhydride, poly ether, polyurea, polyurethane, polyketone, and fluoropolymer.

25. The method of claim 23, wherein the ECM material is mixed with the natural or synthetic polymer composition prior to or during gelation of the ECM material.

26. The method of claim 24, wherein the pre-gel is mixed with fibrin and fibrinogen and is gelled while the fibrin is cross-linked with the fibrinogen.

27. An ECM composition comprising decellularized, acid-protease- digested vaginal tissue, having a pH of from 6.8 to 7.8.

28. The ECM composition of claim 27, wherein the composition is a gel and as compared to acid-protease-digested porcine small intestine submucosa, the gel comprises longer fibers and at least 50% lower FGF-1 and / or FGF-2 content, and optionally has increased HB-EGF (Heparin Binding EGF Like Growth Factor) content and / or lower content of one or more of Angiopoietin 2; Endostatin; IGFBP1 (Insulin Like Growth Factor Binding Protein 1); PTX3 (Pentraxin 3); Prolactin; Serpin B5; and / or TIMP4 (TIMP Metallopeptidase Inhibitor 4), and optionally has at least 50% lower FGF-1 and / or FGF-2 content, increased HB-EGF (Heparin Binding EGF Like Growth Factor) content, and lower content of Angiopoietin 2; Endostatin; IGFBP1 (Insulin Like Growth Factor Binding Protein 1); PTX3 (Pentraxin 3); Prolactin; Serpin B5; and TIMP4 (TIMP Metallopeptidase Inhibitor 4).

29. The composition of claim 27 or claim 28, wherein the acid-protease- digested vaginal tissue is not dialyzed or chemically crosslinked.

30. The composition of claim 27, wherein the ECM composition is a lyophilized powder.

31. A method of preparing an extracellular matrix (ECM) material, comprising: a. exposing vaginal tissue to at least one freeze-thaw cycle; b. disinfecting the tissue, optionally with at least one antibiotic and / or antimycotic composition, thereby producing a decellularized ECM material; c. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; d. comminuting the lyophilized ECM material; e. partially or completely solubilizing the lyophilized ECM material with an acid protease to produce solubilized ECM; and f. optionally lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

32. The method of claim 31 , further comprising neutralizing the lyophilized, solubilized ECM to produce an ECM pre-gel.

33. The method of claim 31, further comprising including one or more washing steps prior to the step of comminuting the lyophilized ECM material.

34. The method of claim 33, wherein the one or more washing steps comprises washing the tissue or material with phosphate-buffered saline, saline, and / or water.

35. The method of claim 31, wherein the ECM material is prepared without a dialysis step or a crosslinking step.

36. The method of claim 31, wherein the acid protease is pepsin and / or trypsin.

37. The method of claim 31, wherein the lyophilized ECM material is solubilized with an acid protease in a solution having a pH of from 1 to 4, from 1 to 2, or 2.0 ± 0.3.

38. The method of claim 31, comprising dispersing the ECM material in a natural or a synthetic polymer composition.

39. The method of claim 38, wherein the natural or a synthetic polymer composition is one or more of: a second ECM material, fibrin, collagen, polyester (PE), polyurethane (PU), poly(ester urethane) urea (PEUU), poly(ether ester urethane) urea (PEEUU), poly (ester carbonate urethane)urea (PECUU), poly (carbonate urethane)urea (PCUU) copolymer, polyolefin (poly alkene), polycarbonate, poly anhydride, poly ether, polyurea, polyurethane, polyketone, and fluoropolymer.

40. The method of claim 38, wherein the ECM material is mixed with the natural or synthetic polymer composition prior to or during gelation of the ECM material.

41. The method of claim 39, wherein the pre-gel is mixed with fibrin and fibrinogen and is gelled while the fibrin is cross-linked with the fibrinogen.

42. A method of preparing an extracellular matrix (ECM) material, comprising: a. incubating vaginal tissue in trypsin;incubating the vaginal tissue in a solution comprising Triton-X-100; c. incubating the vaginal tissue in a solution comprising sodium deoxy cholate; c. incubating the vaginal tissue in a composition comprising ethanol and / or peracetic acid, thereby producing a decellularized ECM material; d. lyophilizing the decellularized ECM material, thereby producing a lyophilized ECM material; e. comminuting the lyophilized ECM material; f. partially or completely solubilizing the comminuted, lyophilized ECM material with pepsin to produce solubilized ECM; and g. lyophilizing the solubilized ECM to produce lyophilized, solubilized ECM.

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