Vaginal extracellular matrix hydrogel and methods for treating vaginal morbidities

Abstract

Compositions and methods of treatment for vaginal morbidities are disclosed herein. Methods for the manufacture of vaginal extracellular matrix hydrogel compositions are disclosed.

Description

VAGINAL EXTRACELLULAR MATRIX HYDROGEL AND METHODS FOR TREATING VAGINAL MORBIDITIESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 557,161, filed February 23, 2024, which is incorporated herein by reference.GOVERNMENT SPONSORSHIP

[0002] This invention was made with government support under grant R01HD102184 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present invention relates to compositions and methods of treatment vaginal morbidities.BACKGROUND

[0004] Genitourinary syndrome of menopause (GSM) refers to the pathological changes in the vulvovaginal and lower urinary tract tissues as a result of the hypoestrogenemia in perimenopausal and menopausal women.1Up to 85% of women over 40 years of age are affected by some form of GSM, with 30-60% of women with GSM suffering from vaginal pruritis and irritation.1Not surprisingly, women with GSM report that this condition negatively affects their sexual activities, interferes with their enjoyment of life and sleep, and hinders traveling, work, and athletic and social activities.2Given that an estimated 47 million women become menopausal every year3and that the expected lifespan is continuously increasing, this population is rapidly expanding.4As such, GSM represents a major public health issue, making it imperative to design accessible and effective treatments for this morbid condition.

[0005] GSM is initiated by the hypoestrogenic state consequent to the cessation of ovarian function, defined as menopause.1Estrogen deprivation results in decreased proliferation and differentiation of vaginal epithelial cells, leading to the disappearance of vaginal rugae and leaving a thin layer of basal epithelium.5,6Further pathological changes include diminished vascularization, reduced smooth muscle content and contractility within the fibromuscularis, andbiochemical and biomechanical alterations of the extracellular matrix (ECM).7,8Lastly, it is known that immune cells of the female reproductive tract are responsive to estrogen; in the hypoestrogenic state, increased expression of pro-inflammatory cytokines and a reduction in important immune populations, including T-regulatory cells, are hallmarks of the GSM phenotype.9The immune system is intricately involved with ECM remodeling,10and both immune activation and ECM remodeling within vaginal tissues are impacted by estrogen deprivation in menopause.11

[0006] Considering the complex cascade of events consequent to the hypoestrogenic state, none of the existing treatments mitigate pathological alterations in every vaginal layer when used alone.2Low-dose vaginal estrogen therapy is the gold-standard GSM treatment, and it is highly effective at restoring epithelium through estrogen-sensitive immunomodulation pathways; however, it fails to improve the fibromuscular phenotype.12Furthermore, despite the benefits and established safety of low dose vaginal estrogen, a number of patients and even some clinicians still reject this treatment because of the concerns of increased risk of hormone-sensitive cancers, especially with the cream formulation of vaginal estrogen.13Importantly, menopause can alter expression of estrogen receptors in genitourinary tissues, further compromising the effectiveness of estrogen-based therapeutics for GSM.14Alternatively, laser therapy has shown some efficacy for repairing the fibromuscularis, but side effects of epithelial burns, scarring, and chronic pain incited an FDA warning against this treatment.15 17Importantly, only 35% of women suffering from GSM are satisfied with their treatment, whether prescribed or over-the-counter.2

[0007] Some biomaterials have been used for treatment of GSM or are under investigation. For example, hyaluronic acid gels have been used to alleviate vaginal dryness and discomfort, though it is inferior to vaginal estrogen and does not exert a regenerative effect on vaginal tissues.18Additionally, intravaginal administration of a recombinant collagen-derived adhesion protein was also explored, and demonstrated increased epithelial proliferation and differentiation following treatment in a rat model of menopause.19It has also been previously demonstrated that collagen- based biomaterials are inferior in inducing regeneration compared to tissue-specific ECM hydrogels.20Decellularized ECM hydrogels have been extensively investigated as pro- regenerative scaffolds in various pre-clinical tissue engineering applications21as well as demonstrated safety and feasibility in Phase I Clinical trial (clinicaltrial.gov NCT02305602) for the treatment of myocardial infarction.22Decellularized ECM hydrogels have demonstrated tissue regenerative capabilities in numerous injury and disease models through mechanisms of cellularrecruitment, increased cellular proliferation and differentiation, immune modulation, neovascularization, and ECM remodeling.21However, these applications have mainly utilized needle based injections into tissue rather than topical applications in hollow organs, and none have been applied intravaginally.SUMMARY OF THE INVENTION

[0008] Compositions and methods of treatment for vaginal morbidities are disclosed herein.

[0009] In one aspect, a biocompatible material having decellularized extracellular matrix derived from vaginal tissue is provided. In aspects, the vaginal tissue is selected from the group consisting of human vaginas, porcine vaginas, bovine vaginas, goat vaginas, mouse vaginas, rat vaginas, rabbit vaginas, and chicken vaginas. In some aspects, the decellularized vaginal extracellular matrix is in an injectable liquid or solution form. In some aspects, the decellularized vaginal extracellular matrix is in an injectable gel. In some aspects, the decellularized vaginal extracellular matrix is in an injectable particulate.

[0010] In another aspects, a method of treating vaginal morbidities is provided, including vaginal atrophy (thinning of the epithelial layer, loss of vaginal rugae, loss of smooth muscle layer), insufficient vaginal lubrication, fibrosis, scarring / adhesions, stenosis, shortening, pallor, firmness, telangiectasias, fragility, exposure of the implanted synthetic grafts, granulation tissue due to reaction to the implanted synthetic grafts or sutures, bleeding, ulcerations, and alterations in vaginal mechanical properties, such as decreased elasticity, increased stiffness, comprising administering to the subject an effective amount of a composition comprising decellularized extracellular matrix (ECM).

[0011] In some aspects, the decellularized extracellular matrix is derived from vaginal tissue. In some aspects, the decellularized extracellular matrix is derived from a tissue containing striated muscle or smooth muscle. In some aspects, the decellularized extracellular matrix is derived from small intestine submucosa, bladder, uterus, heart, vagina, liver, lung, or blood vessels.

[0012] In some aspects, the composition is coated on an implant. In some aspects, the composition is delivered as a liquid or a powder. In some aspects, the composition transitions to a gel form after delivery. In some aspects, the composition degrades within one day to three months following delivery. In some aspects, the extracellular matrix is lyophilized and rehydrated prior to injection or topical application. In some aspects, the delivery is transvaginal. In some aspects, thedelivery is intravaginal. In some aspects, the composition is a wash, a liquid, a particulate, or a hydrogel applied topically. In some aspects, the composition is injected through a small gauge needle into vaginal tissue.

[0013] In some aspects, the composition further comprises an additional component selected from the group consisting of proteins, peptides, polypeptides, exogenously added cells, autologous cells, nutrients, synthetic polymers, survival promoting additives, proteoglycans, glycosaminoglycans, therapeutic agents, and a combination thereof. In some aspects, the composition further comprises cellular growth factors. In some aspects, the composition further comprises stem cells. In some aspects, the composition further comprises growth factors, stem cells or autologous cells, and a therapeutic agent.

[0014] In a further aspect, a method of formulating an extracellular matrix (ECM) composition is provided. The method includes the steps of isolating a tissue sample; decellularizing the tissue sample; lyophilizing the tissue sample; milling the tissue sample; and enzymatically digesting the tissue sample; where the ECM composition is effective to treat a vaginal morbidity in a subject. In aspects, the tissue sample comprises vaginal tissue selected from the group consisting of human vaginas, porcine vaginas, bovine vaginas, goat vaginas, mouse vaginas, rat vaginas, rabbit vaginas, and chicken vaginas. In aspects, the vaginal morbidity is selected from vaginal atrophy (thinning of the epithelial layer, loss of vaginal rugae, loss of smooth muscle layer), insufficient vaginal lubrication, fibrosis, scarring / adhesions, stenosis, shortening, pallor, firmness, telangiectasias, fragility, exposure of the implanted synthetic grafts, granulation tissue due to reaction to the implanted synthetic grafts or sutures, bleeding, ulcerations, and alterations in vaginal mechanical properties, such as decreased elasticity, increased stiffness. In aspects, the effective amount of the ECM composition comprises a concentration of greater than about 6 mg of ECM per mL of the ECM composition, and wherein the effective amount of the extracellular matrix (ECM) composition comprises a vaginal extracellular matrix hydrogel effective to treat a vaginal morbidity.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1. Production of a porcine vaginal tissue derived ECM hydrogel (vECM). Whole porcine vaginas were cleaned and frozen (Panel A) before being minced with a commercial meat grinder (Panel B) then spun in SDS to decellularize the tissue Panel (C). Decellularized tissue waslyophilized (Panel D) and milled to a fine and unform powder (Panel E). Decellularized vECM powder was partially enzymatically digested with pepsin (Panel F) then pH and salt balanced before incubating at 37°C to form a stable hydrogel (G).

[0016] Figure 2. Characterization of vECM structure, composition, and material properties. Vaginal samples were taken from fresh tissue (Panel A) or tissue after either 1 (Panel B), 2 (Panel C), or 3 (Panel D) days of decellularization. Tissue samples were cryosectioned and stained with hematoxylin and eosin to demonstrate removal of nuclear material (purple) and retention of ECM. (Panel E) Porcine vaginal tissue and decellularized vECM were analyzed via ECM targeted proteomics using QConCAT (n=3 / group). (Panel F) 6 and 8 mg / mL vECM preparations (n=3 / group) underwent complex viscosity testing with a parallel plate rheometer, demonstrating shear thinning properties for both groups, and an increased viscosity in the 8 mg / mL group at lower shear rates (black arrows). (Panel G) vECM was prepared at 6 and 8 mg / mL concentrations, and n=3 wells per concentration underwent a turbidity assay, in which OD was read at 405 nm for two hours or until equilibrium. (Panel H) The time to 50% equilibrium OD was determined for 6 and 8 mg / mL, demonstrating a significantly lower time for the 8 mg / mL group.

[0017] Figure 3. Evaluation of vECM efficacy in restoring vaginal epithelium in menopausal rats. (Panel A) Experimental timeline to assess therapeutic efficacy of intravaginal administration of vECM, alongside saline and collagen, in a rat model of surgical menopause via ovariectomy. After treatment, vaginal tissues were isolated, sectioned, and stained with hematoxylin and eosin to assess vaginal epithelial thickness. Representative images are shown for healthy (unperturbed) controls (Panel B) and ovariectomized rats treated with saline (Panel C), collagen (Panel D), 6 mg / mL vECM (Panel E), or 8 mg / mL vECM (Panel F). Data for epithelial thickness were averaged per animal and compared among groups with a one-way ANOVA and Tukey’s pairwise comparisons (Panel G).

[0018] Figure 4. Characterization of vaginal epithelial stem cells in vECM treated animals. Vaginal tissue sections were stained against nuclei (DAPI, blue), an epithelial stem cell transcription factor (p63, magenta), and an epithelial membrane marker (filaggrin, green) to identify epithelial stem cells. Representative immunofluorescent images are shown for healthy (unperturbed) controls (Panel A) and ovariectomized rats treated with saline (Panel B), collagen (Panel C) or 8 mg / mL vECM (Panel D). Vaginal epithelium was assessed for nuclei density (Panel E), p63+ vaginal epithelial stem cell nuclei density (Panel F), and percentage of nuclei within theepithelium that were p63+ vaginal epithelial stem cells (Panel G). Data were compared among groups with a one-way ANOVA and Tukey’s pairwise comparisons.

[0019] Figure 5. Evaluation of vECM efficacy in restoring vaginal smooth muscle in menopausal rats. Vaginal tissue sections were stained against nuclei (DAPI, blue) and smooth muscle (aSMA, yellow). Representative immunofluorescent images are shown for healthy (unperturbed) controls (Panel A) and ovariectomized rats treated with saline (Panel B), collagen (Panel C), 6 mg / mL vECM (Panel D), or 8 mg / mL vECM (Panel E). Data for smooth muscle thickness were averaged per animal and compared among groups with a one-way ANOVA and Tukey’s pairwise comparisons (Panel F).

[0020] Figure 6. Assessment of vaginal smooth muscle cell proliferation following treatment. Vaginal tissue sections were stained against nuclei (DAPI, blue), smooth muscle actin (aSMA, green), and a nuclear marker of proliferating cells (Ki67, cyan). Representative immunofluorescent images are shown for healthy (unperturbed) controls (Panel A) and ovariectomized rats treated with saline (Panel B), collagen (Panel C), or 8 mg / mL vECM (Panel D). Groups were assessed for Ki67+ nuclei density within the smooth muscle layer (Panel E), and proportion of Ki67+ nuclei over total nuclei within the smooth muscle layer (Panel F). Data were averaged per animal and compared among groups with a one-way ANOVA and Tukey’s pairwise comparisons.

[0021] Figure 7. vECM at two doses and collagen control demonstrate similar retention in the vaginal lumen and fibromuscularis. Two weeks following ovariectomy procedures, rats were treated with a single 500 pL intravaginal administration of fluorescently pre-labeled collagen (Panel A), 6 mg / mL vECM (Panel B), or 8 mg / mL vECM (Panel C). Tissue sections were harvested at 1, 2, or 3 days post-administration. Representative tissue sections are shown for each group, in which fluorescently tagged biomaterial is shown in cyan, and tissues were stained against cell nuclei (DAPI, blue). Tissue sections were assessed for material retention by assessing the average area of material throughout the tissue (Panel D). Additionally, material from all groups demonstrated material in the vaginal fibromuscularis; the frequency of this observation was reported as percentage of sections (Panel E). Sections were stained against macrophage marker (CD68, red), which was observed colocalized in both the vaginal lumen and fibromuscularis in all groups (Panel F).

[0022] Figure 8. Assessment of macrophage infiltration in vaginal tissues after various treatments. Vaginal tissue sections were stained against nuclei (DAPI, blue), a pan-macrophagemembrane marker (CD68, red), and an M2 macrophage membrane marker (CD163, green). Representative immunofluorescent images are shown for healthy (unperturbed) controls (Panel A) and ovariectomized rats treated with saline (Panel B), collagen (Panel C), or vECM 8 mg / mL (Panel D). Groups were assessed for nuclei density within the lamina propria and muscularis (Panel E), proportion of CD68+ nuclei, indicating macrophages (Panel F), and proportion of CD163+ nuclei, indicating anti-inflammatory M2 macrophages (Panel G). Data were averaged per animal and compared among groups with a one-way ANOVA and Tukey’s pairwise comparisons.DETAILED DESCRIPTION

[0023] More than half of menopausal women experience vaginal pruritis, dryness, pain, and discomfort associated with GSM. Unfortunately, the only effective treatment, vaginal low dose estrogen, is not effective for restoring fibromuscular phenotype in GSM, and some reject it out of fear of hormone associated cancer risks despite existing evidence to the contrary.13Thus, a nonhormonal, acellular, and easily manufactured tissue regenerative biomaterial has been developed as an alternative treatment for vaginal atrophy associated with GSM.

[0024] Acellular vaginal scaffolds for vaginal reconstruction32or 3D printed vaginal tissue analogues have been explored, incorporating gelatin and sodium alginate,33,34but no acellular vaginal tissue-derived ECM hydrogel has been developed, the physical properties of which make this material well suited for topical intravaginal administration. Additionally, ECM hydrogels have not been previously examined for this application, which is uniquely challenging as the intravaginal environment, owing to constant motion in an open cavity, does not allow for true gelation of the material. In the context of other ECM biomaterials, optimal decellularization conditions for vaginal tissue differed considerably from that of striated muscle35and small intestinal submucosa36, while sharing similarities with some protocols for skin decellularization.37As the vagina has a relatively low cellular content and is largely composed of ECM, vaginal tissue required gentler decellularization and a more thorough water rinse to preserve ECM structure and sufficiently remove residual detergent.

[0025] To assess the therapeutic potential of vECM, the treatment regimen of locally delivered vaginal estrogen creams were mirrored, which are applied once daily for a period of two weeks, after which 2-3 applications per week are used for maintenance.38Locally delivered estrogen, the current clinical gold standard of treatment for vaginal atrophy associated with GSM, activatesestrogen-sensitive pathways in the vaginal tissues to promote epithelial growth and maturation index,39though neither local nor systemic estrogen treatment effectively improves the fibromuscular layer.29-40vECM, at the higher dose, not only significantly improved epithelial thickness and returned epithelial phenotype to a healthier state, e.g., healthier multilayers striated epithelium, but additionally impacted vaginal smooth muscle thickness and proliferation. For both of these metrics, a dose-dependent response in regeneration was observed; an 8 mg / mL formulation of vECM performed better than a 6 mg / mL formulation, and the invention provides that even higher concentrations can deliver improved results.

[0026] The impact of vECM on the subepithelial tissues are likely due to the observed vECM localization in lamina propria and fibromuscularis, where the material colocalized with cell nuclei in all observations, oftentimes with co-staining of macrophage membrane markers. vECM and collagen biomaterial components are too large to passively bypass the vaginal epithelium at acute timepoints. It is therefore believed that cells may be trafficking vECM and collagen through the epithelium and into subepithelial tissues. It has been demonstrated that macrophages can uptake synthetic biomaterials, which then facilitate intracellular modulation of macrophage activity.41Overall, cellular interactions and localization were consistent between all biomaterial groups, suggesting that the superior therapeutic efficacy of the at least 8 mg / mL vECM may be due to the increased concentration of bioactive ECM components. Decellularized ECM components have been shown to polarize macrophages to an M2 like phenotype and stimulate endogenous tissue repair mechanisms in a variety of applications, such as spinal cord injury,42wound healing,43and osteochondral defects.44Furthermore, various studies have explored the pivotal role of macrophages in epithelial remodeling and regeneration in nasal,45colonic,46and lung47epithelia as well as smooth muscle regeneration.48-49As described herein, after 2 weeks of treatment, the 8 mg / mL vECM group demonstrated significant recruitment of macrophages and polarization to M2 phenotype in vaginal tissues; this indicates that the tissue regeneration observed in the epithelium and fibromuscularis may result from pro-regenerative immune modulation.

[0027] A vaginal tissue-derived decellularized ECM hydrogel as described herein was therefore developed and it was discovered that when delivered intravaginally as a topical application, it improved epithelial thickness, epithelial phenotype, smooth muscle thickness, and smooth muscle proliferation, and recruited macrophages, which were polarized to a pro- regenerative phenotype. Furthermore, it was discovered that biomaterial formulations are wellretained in the vagina and are even trafficked to subepithelial tissues following intravaginal topical administration. The development and application of this novel acellular biomaterial therapeutic for vaginal atrophy associate with GSM is therefore described herein in greater detail.

[0028] Various aspects and embodiments of the disclosure are provided by the following description. Before describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. It is also to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as such.

[0029] Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and uses thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0031] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology,cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, and periodic updates); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22th ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012).

[0033] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a fusion protein, a pharmaceutical composition, and / or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the fusion protein, pharmaceutical composition and / or method.

[0034] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0035] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a fusion protein, pharmaceutical composition, and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel character! stic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0036] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0037] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0038] It is understood that aspects and embodiments of the present disclosure described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments.

[0039] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and / or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0040] The present disclosure provides decellularized extracellular matrix (ECM) compositions and methods for treatment of vaginal morbidities.

[0041] In embodiments, the ECM can be prepared through methods such as described herein and in Ungerleider, et al. JACC: Basic to Translational Science, Vol 1, No. 1-2 (2016), which is incorporated by reference in its entirety. In embodiments, the tissue is first decellularized, leaving only the extracellular matrix such as disclosed in U.S. Patent Publication US2013 / 0251687, for example, which is incorporated by reference in its entirety. The matrix is then lyophilized, ground or pulverized into a fine powder, solubilized with pepsin or other enzymes, and subsequently neutralized and buffered as previously reported. ECM can be lyophilized, and stored in sterile containers. ECM can be resuspended to appropriate / physiological concentration for injection or topical application.

[0042] In embodiments, the effective amount of the ECM composition comprises a concentration of greater than about 6 mg of ECM per mL of the ECM composition, or about 8 mg or greater of ECM per mL of the ECM composition.

[0043] In embodiments, the decellularization step comprises about 3 days of 0.1% SDS decellularization. In embodiments, the decellularization step further comprises about 3 days of rinsing to remove SDS.

[0044] In embodiments, a composition having decellularized ECM derived from vaginal tissue or other suitable tissues is provided, which may be referred to herein as vECM. The composition may be injectable. The composition may be formulated into a powder or particulate. In embodiments, the composition may be formulated to be in liquid form at room temperature, typically 20°C to 25°C, and in gel form at a temperature greater than room temperature, or greater than 35°C. In embodiments, the composition may be configured to be delivered to a tissue parenterally, such as through a small gauge needle (e.g., 27 gauge or smaller). In embodiments, the composition may be suitable for direct implantation into a patient. The composition may be formulated either in a dry or hydrated form to be placed on or near injured or missing tissue.

[0045] After adjusting concentration, the ECM composition can be lyophilized and stored frozen (e.g. -20C, -80C) for at least 3 months. The ECM composition can then be rehydrated with sterile water prior to injection or topical application.

[0046] The ECM composition can be directly injected, such as with a 25, 27, 30 gauge or smaller needle, intravaginal applicator, or intravaginal drug releasing devices or pessaries.

[0047] The ECM composition gel can be crosslinked with glutaraldehye, formaldehyde, bis- NHS molecules, or other crosslinkers. The ECM composition can be combined with cells,peptides, proteins, DNA, drugs, nutrients, survival promoting additives, proteoglycans, and / or glycosaminolycans. The ECM composition can be combined and / or crosslinked with a synthetic polymer. The ECM composition can be used alone or in combination with above described components for endogenous cell ingrowth, angiogenesis, and regeneration. The ECM composition can be use alone or in combination with above described components as a matrix to change mechanical properties of the tissue. The ECM composition can be delivered with cells alone or in combination with above described components for regenerating damaged tissue.

[0048] In embodiments, the present disclosure provides that the ECM is combined with cells, peptides, proteins, DNA, drugs, nanoparticles, antibiotics, growth factors, nutrients, exosomes and extracellular vesicles, survival promoting additives, proteoglycans, and / or glycosaminoglycans.

[0049] The vECM may be combined with thickening agents (i.e. carbomer, hydroxyethyl cellulose) to alter rheological properties of the material. The vECM may be derived from suitable tissue, which could be vaginal, uterine, small intestine submucosa, bladder, lung, or vascular for example.

[0050] The vECM may be configured to coat surfaces to culture vaginal epithelial cells or other cell types related to vaginal tissue, such as fibroblasts, smooth muscle cells, vascular cells. The vECM can be placed into tissue culture plates or wells to for a gel used for cell culture. The vECM can be provided in the shape of a mold, or the matrix may be molded to the shape of the substrate.

[0051] Cells may also be cultured on an adsorbed matrix, which would involve incubating tissue culture plates with decellularized vECM material, then removing said material. Cells may then be cultured, including vaginal epithelial cells or other cells relevant to vaginal tissue repair.

[0052] In embodiments, the present disclosure provides that the composition of infusible extracellular matrix is derived from human, animal, embryonic, and / or fetal tissue sources.

[0053] In embodiments, the composition is a liquid form of vaginal matrix can assembly into a fibrous scaffold upon injection in vivo. The material can also be processed into a lyophilized form that requires only sterile water, phosphate buffered saline (PBS), or saline to resuspend prior to injection or topical application, which can provide ease of storage and use in a clinical setting.

[0054] In embodiments, the composition further includes cells, drugs, proteins, or polysaccharides. In embodiments, the composition is delivered as a liquid, and in many instances,the composition may transition to a gel form after delivery. In embodiments, the composition is delivered as a powder.

[0055] In embodiments, the composition includes native proteins. In embodiments, the composition includes native peptides. In embodiments, the composition includes native glycosaminioglycans. In embodiments, the composition also includes non-naturally occurring factors that recruit cells into the composition, encourage growth, or prevent infection. In embodiments, the composition including decellularized ECM derived from vaginal tissue (vECM) retains native glycosaminoglycans. In embodiments, the composition includes naturally occurring factors that recruit cells into the composition, encourage growth, or prevent infection.

[0056] In embodiments, the composition further includes a population of exogenous or autologous therapeutic cells. The cells may be stem cells or other precursors of vaginal cells or other cell types.

[0057] In embodiments, the composition further includes a therapeutic agent, and as such, is configured as a drug delivery vehicle.

[0058] In embodiments, a method of producing a composition with decellularized ECM derived from vaginal or other tissue is provided, the method including the steps of: obtaining from a subject a vaginal or other suitable tissue sample having an extracellular matrix and non- extracellular matrix components; processing vaginal or other tissue sample to remove the non- extracellular matrix component to obtain decellularized vaginal or other tissue extracellular matrix and extracellular proteins and polysaccharides; and sterilizing the decellularized vaginal or other tissue extracellular matrix. In embodiments, the method is performed aseptically without sterilization. In embodiments, the method further includes the step of lyophilizing and grinding up the decellularized ECM. In embodiments, the method further includes the step of enzymatically treating, solubizing, or suspending the decellularized ECM. In embodiments, the decellularized ECM is digested with pepsin at a low pH.

[0059] In embodiments, the method further includes the step of suspending and neutralizing the decellularized ECM in a solution. In embodiments, the solution is a PCS or saline solution, which can be injected through a high gauge needled into the desired tissue or organ. In embodiments, the composition is formed into a gel at body temperature. In embodiment, the composition further includes cells, drugs, proteins, or other therapeutic agents that can be delivered within or attached to the composition before, during, or after gelation.

[0060] In embodiments, the composition may be used for gel therapy. The composition is neutralized and brought to an appropriate concentration using PBS or saline. In embodiments, the solution can then be injected into the injured tissue or a tissue in need. The needle size may be, without limitation, a 22 gauge, 23 gauge, 24 gauge, 25 gauge, 26 gauge, 27 gauge, 28 gauge, 29 gauge, 30 gauge, or smaller. In embodiments, the needle size through which the solution is injected is a 27 gauge needle.

[0061] In embodiments, ECM solution or gel can be injected into the injured tissue or other relevant tissue in need, alone or in combination with above-described components for endogenous cell ingrowth, angiogenesis, and regeneration. In embodiments, the ECM or ECM liquid can be sprayed on or into injured tissue or other relevant tissue in need in need, alone or in combination with above-described components for endogenous cell ingrowth, angiogenesis, and regeneration. In embodiments, the composition can also be used alone or in combination with above-described components as a matrix to change mechanical properties of the vagina or other relevant tissues and / or to restore mass and function.

[0062] In embodiments, when delivering a composition that comprises the decellularized vaginal ECM and exogenous cells, the cells can be from cell sources for treating certain diseases, such as sources for treating vaginal morbidities, that include allogenic, xenogenic, or autogenic sources. Accordingly, embryonic stem cells, fetal or adult derived stem cells, induced pluripotent stem cells, vaginal progenitors, fetal and neonatal vaginal cells, mesenchymal cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, hematopoetic stem cells, bone marrow-derived progenitor cells, skeletal cells, macrophages, adipocytes, and autotransplanted expanded vaginal cells can be delivered by a composition herein. In some instances, cells herein can be cultured ex vivo and in the culture dish environment differentiate either directly to vaginal cells, or to cells that can become vaginal cells. The cultured cells are then transplanted into the mammal, either with the composition or in contact with the scaffold and other components. Adult stem cells are yet another species of cell that can be part of a composition herein. Adult stem cells are thought to work by generating other stem cells (for example those appropriate to vaginal tissue) in a new site, or they differentiate directly to a vaginal cells in vivo. They may also differentiate into other lineages after introduction to organs, such as the vagina.

[0063] In embodiments, the composition may also include cells, drugs, proteins, or other biological material such as, but not limited to, erythropoietin (EPO), stem cell factor (SCF),vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), cartilage growth factor (CGF), nerve growth factor (NGF), keratinocyte growth factor (KGF), skeletal growth factor (SGF), osteoblast-derived growth factor (BDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), cytokine growth factor (CGF), stem cell factor (SCF), platelet-derived growth factor (PDGF), endothelial cell growth supplement (EGGS), colony stimulating factor (CSF), growth differentiation factor (GDF), integrin modulating factor (IMF), calmodulin (CaM), thymidine kinase (TK), tumor necrosis factor (TNF), growth hormone (GH), bone morphogenic proteins (BMP), matrix metalloproteinase (MMP), tissue inhibitor matrix metalloproteinase (TIMP), interferon, interleukins, cytokines, integrin, collagen, elastin, fibrillins, fibronectin, laminin, glycosaminoglycans, hemonectin, thrombospondin, heparan sulfate, dermantan, chondrotin sulfate (CS), hyaluronic acid (HA), vitronectin, proteoglycans, transferrin, cytotactin, tenascin, and lymphokines.

[0064] In embodiments, vECM is able to be delivered through a catheter or needle for treatment of a malady because ECM is shear-thinning. In embodiments, the catheter or needle can be up to 3 to 5 m in length and have an diameter of 25 G or smaller into vaginal or cervical tissues. The vECM may be administered “topically” or intravaginally into the vaginal lumen as a wash or as a hydrogel. The vECM may also be implanted in vaginal tissue. The vECM may be a gel within 30 minutes after delivery to in vivo tissue, or it may comprise a digestive enzyme to facilitate gelation above 20, 25, 30, or 35 °C and in less than 30, 20, 10, 5, or 1 minute.

[0065] In embodiments, an effective amount of vECM delivered to a subject includes a concentration of vECM about 1-20 mg ECM to mL of total product. In embodiments, an effective amount of vECM delivered to a subject includes a concentration of ECM about 6-20 mg vECM to mL of total product. In embodiments, an effective amount of vECM delivered to a patient includes a concentration of vECM about 6-12 mg vECM to mL of total product. In embodiments, an effective amount of ECM delivered to a subject includes a concentration of ECM greater than 6 mg vECM to mL of total product, or about 8 mg vECM to mL of total product, or greater concentration.

[0066] In embodiments, vECM is delivered in an effective amount to a subject once, or is delivered to a subject multiple times. In embodiments, vECM is delivered to a subject on a schedule or in multiple doses, for example, once a day, once a week, three times a week, once a month, once a year or more or less frequently.

[0067] In embodiments, ECM is delivered at different time courses throughout a subject’s disease as is most appropriate, for example, immediately post-injury, infection, or diagnosis, or additionally, about one hour, several hours, one day, one week, one or more months, or one or more years after injury, infection, or diagnosis.

[0068] In embodiments, the injection, topical application, or implantation of said ECM composition repairs damage to the vagina sustained by said subject. In embodiments, the injection, topical application, or implantation of said composition reduces scar formation in said subject. As used herein, the effective amount can be an amount that reduces vaginal morbidity in the area of the injection, topical application, implantation, or treated tissue of the subject. In embodiments, the effective amount is an amount that increases vaginal regeneration. In embodiments, the effective amount is an amount that increases vaginal function.

[0069] As used herein, “patient” or “subject” means a human or animal subject to be treated.

[0070] As used herein the term “pharmaceutical composition” refers to pharmaceutically acceptable compositions, wherein the composition comprises a pharmaceutically active agent, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination of pharmaceutically active agents and carriers.

[0071] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where one or more active compounds and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances, the combination partners show a cooperative, e.g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the activeingredients, e g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0072] As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and / or non-human mammals.

[0073] As used herein the term “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which demethylation compound(s), is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0074] As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if diseasepathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0075] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from happening) or stopped (e.g. terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0076] As used herein, and unless otherwise specified, the terms “prevent,” “preventing” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term “prevention” may be interchangeably used with the term “prophylactic treatment.”

[0077] As used herein, and unless otherwise specified, a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0078] In embodiments, the invention provides methods of preventing or treating a vaginal morbidity in a subject, the method comprising administering to the subject an effective amount of an extracellular matrix (ECM) composition. In embodiments, the ECM composition is derivedfrom vaginal tissues. In embodiments, the ECM composition comprises a vaginal extracellular matrix (vECM) hydrogel.

[0079] In embodiments, the invention provides a method of treating vaginal morbidities, including vaginal atrophy (thinning of the epithelial layer, loss of vaginal rugae, loss of smooth muscle layer), insufficient vaginal lubrication, vaginal burning, vaginal pruritis, vaginal pain, dyspareunia, fibrosis, scarring / adhesions, stenosis, shortening, pallor, firmness, telangiectasias, fragility, exposure of the implanted synthetic grafts, granulation tissue due to reaction to the implanted synthetic grafts or sutures, bleeding, vaginal epithelial abnormalities, ulcerations, and alterations in vaginal mechanical properties, such as decreased elasticity, increased stiffness, comprising administering to a person in need thereof an effective amount of a composition comprising decellularized extracellular matrix derived from vaginal tissue (vECM).

[0080]

[0081] In embodiments, the ECM composition is injected at the site of the vaginal injury. In embodiments, the effective amount of the ECM composition comprises a concentration of about 1 mg to about 20 mg of ECM per m of the ECM composition or greater than 6 mg to about 12 mg, or about 8 mg ECM to mL of ECM composition.

[0082] In embodiments, the invention provides methods of using a vaginal extracellular matrix (vECM) composition, the method comprising: administering an effective amount of the vECM composition to a subject; wherein administering the vECM composition is effective to treat vaginal injury in the subject. In embodiments, the ECM composition comprises a vaginal extracellular matrix (vECM) hydrogel. In embodiments, the vECM composition is injected at the site of the vaginal injury, infused via a catheter, administered intravenously, or administered via intravascular infusion. In embodiments, the effective amount of the vECM composition comprises a concentration of about 1 mg to about 20 mg of vECM per mL of the vECM composition. In embodiments, the effective amount of the vECM composition is administered to the subject once. In embodiments, the effective amount of the vECM composition is administered to the subject periodically at an interval of once per day, once per week, once per month, or once per year. In embodiments, administering the effective amount of the vECM composition is effective to promote vaginal tissue regeneration, engraftment, growth, volume, bulk, or function.

[0083] In embodiments, the invention provides vaginal extracellular matrix (vECM) compositions, and methods of formulating such vECM compositions, the method comprising:isolating a tissue sample; decellularizing the tissue sample; lyophilizing the tissue sample; milling the tissue sample; and enzymatically digesting the tissue sample; wherein the ECM composition is effective to vaginal morbidities in a subject, as described herein.EXAMPLES

[0084] It will be understood from the foregoing description that various modifications and changes may be made in the various embodiments of the present disclosure without departing from their true spirit. The description provided herein is intended for the purposes of illustration only and is not intended to be construed in a limiting sense. Thus, while the presently disclosed inventive concepts have been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the presently disclosed inventive concepts be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications, and equivalents are included within the scope of the presently disclosed inventive concepts as defined herein.

[0085] Thus, the examples described herein, which include particular embodiments, will serve to illustrate the practice of the presently disclosed inventive concepts, it being understood that the particulars show are by way of example and for purposes of illustrative discussion of particular embodiments of the presently disclosed inventive concepts only and are presented in the cause of providing what is believed to be a useful and readily understood description of procedures as well as the principles and conceptual aspects of the inventive concepts. Changes may be made in the construction and formulation of the various components and compositions described herein, the methods described herein, or in the steps of the sequence of steps of the methods described herein without departing from the spirit and scope of the presently disclosed inventive concepts.Example 1. Materials and MethodsFabrication and Characterization of Vaginal (vECM) Hydrogel

[0086] Porcine vaginal tissues were acquired from young female pigs sourced from Midwest Research Swine (Collagen Solutions). Several rounds of decellularization optimization were conducted, in which the following conditions were tested: tissue processing method (handchopped or meat grinder), SDS concentration (0.1, 0.5, or 1%), duration of decellularization (3 or 5 days), and duration of water rinse (1 or 3 days). For all decellularization conditions, decellularized ECM was lyophilized, milled, and passed through a #60 fdter. Milled and fdtered vECM underwent pepsin digestion at a 10 mg ECM / mL pepsin solution in 0. IM HC1 for 48 hours.Digested vECM was pH and salt balanced, then incubated at concentrations of 4, 6, or 8 mg / mL at 37°C for 24 hours to test gelation. vECM was screened for residual nuclear material, using PicoGreen (Thermo Fisher) fluorescent assay; residual SDS, using methylene blue assay; and sulfated glycosaminoglycans, using 1,9-dimethylmethylene blue assay.

[0087] ECM specific protein makeup of fresh tissue and decellularized powder were assessed via ECM targeted proteomics (n=3 / group) using quantitative concatemers, described as follows.26

[0088] Sample preparation for proteomic analysis: Two milligrams of lyophilized material from each sample were homogenized in 200 pL / mg of 6 M guanidine hydrochloride (Gnd-HCl), 100 mM ammonium bicarbonate (ABC) at power 8 for 1 minute (Bullet Blender, Model BBX24, Next Advance, Inc.) and vortexed (power 5) at room temperature overnight. Homogenate was spun at 18,000 x g (4°C) for 15 min and the supernatant was collected as the soluble ECM (sECM) fraction. Pellets were then treated with freshly prepared hydroxylamine (HA) buffer (1 M NH2OH-HCI, 4.5 M Gnd-HCl, 0.2 M K2CO3, pH adjusted to 9.0 with NaOH) at 200 pL / mg of the starting tissue dry weight. Samples were homogenized at power 8 for 1 minute and incubated at 45°C with shaking (1000 rpm) for 4 h. Following incubation, the samples were spun for 15 min at 18,000 x g and the supernatant was removed and stored as the insoluble ECM (iECM) fraction at -80°C until further proteolytic digestion. All fractions were subsequently subjected to enzymatic digestion overnight (16 h) at 37°C with trypsin (1:100 enzyme to protein ratio) using a filter aided sample preparation (FASP) approach as previously described30and desalted during Evotip loading.

[0089] LC-MS / MS analysis: Digested peptides were loaded onto individual Evotips following the manufacturer’s protocol and separated on an Evosep One chromatography system (Evosep, Odense, Denmark) using a Pepsep column, (150 pm inter diameter, 15 cm) packed with ReproSil C18 1.9 pm, 120A resin. Samples were analyzed using the instrument default “30 samples per day” LC gradient. The system was coupled to the timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) via the nano-electrospray ion source (Captive Spray, Bruker Daltonics). The mass spectrometer was operated in PASEF mode. The ramp time was set to 100 ms and 10 PASEF MS / MS scans per topN acquisition cycle were acquired. MS and MS / MS spectra were recorded from m / z 100 to 1700. The ion mobility was scanned from 0.7 to 1.50 Vs / cm2. Precursors for data-dependent acquisition were isolated within ± 1 Th and fragmented with an ion mobility-dependent collision energy, which was linearly increased from 20 to 59 eV in positivemode. Low-abundance precursor ions with an intensity above a threshold of 500 counts but below a target value of 20000 counts were repeatedly scheduled and otherwise dynamically excluded for 0.4 min.

[0090] Global proteomic data analysis: Data were searched using MSFragger v3.8 via FragPipe v20.0. Precursor tolerance was set to ±15 ppm and fragment tolerance was set to ±0.08 Da. Data were searched against UniProt restricted to Sus scrofa with added common contaminant sequences (46,291 total sequences). Enzyme cleavage was set to semi-specific trypsin for all samples. Fixed modifications were set as carbamidomethyl (C). Variable modifications were set as oxidation (M), oxidation (P) (hydroxyproline), deamidation (NQ), Gln->pyro-Glu (N-term Q), and acetyl (protein N-terminus). Label free quantification was performed using lonQuant vl.9.8 with match-between-runs enabled and default parameters. Soluble and insoluble ECM fractions were searched separately and merged after database searching. Results were filtered to 1% FDR at the peptide and protein level.

[0091] Mechanical properties: To evaluate vECM mechanical properties, vECM was prepared at 6 and 8 mg / mL concentrations. Optical density was used to evaluate the turbidimetric gel kinetics of vECM. 100 pL of vECM was pipetted into a 96 well plate (n=3 wells / concentration group). The plate was loaded into a preheated (37°C) spectrophotometer, and absorbance was read at 405 nm every minute for two hours or until equilibrium was reached. To measure complex viscosity, 200 pL of vECM (n=3 runs / concentration group) was dispensed on a parallel plate ARG2 rheometer stage (TA Instruments) at 25°C. Gap height was set to 500 pm, and a flow procedure was executed with shear rate ranging from 0.1 - 1000 Hz.Preparation of vECM and Collagen for Intravaginal Administration.

[0092] Two different concentrations of vECM (6 mg / mL or 8 mg / mL) were prepared for intravaginal administration by modulating water and salt (PBS) addition during the resuspension stage. As a non-tissue specific control, collagen 1 derived from rat tail (Coming) was pH and salt balanced and used at a 6.5 mg / mL concentration in order to match the rheological properties (specifically low-shear rate complex viscosity) of vECM at the 8 mg / mL concentration.Intravaginal Administration of Saline, Collagen, and vECM.

[0093] All procedures were approved by the Institutional Animal Care and Use Committee at the University of California San Diego. A well-established bilateral ovariectomy (OVX) rat model of surgical menopause (Charles River) was used. Two weeks after OVX, a duration previouslyestablished to be sufficient to induce changes driven by estrogen deprivation in this model,283- month-old Sprague Dawley rats were anesthetized with 2.5% isoflurane in oxygen for the duration of the procedure and placed in supine position. A 1 mL syringe without needle was loaded with 500 pL of saline, collagen, or vECM at 6 mg / mL or 8 mg / mL concentrations (n=4-8 / group). The animals were left to rest for 10 minutes, after which they were returned to housing and observed until mobile. These treatments were carried out daily for a duration of two weeks. The day after the end of treatment, animals were euthanized, and full-thickness vaginas were harvested, flash frozen, and stored in -80C for subsequent cryosectioning, staining, and analyses. Age-matched non-ovariectomized animals were used as healthy controls (n=3).

[0094] To assess retention and localization of vECM in the vagina, a separate group of OVX rats was used. For all biomaterial groups (vECM 6 mg / mL, vECM 8 mg / mL, and collagen) materials were mixed with an NHS Succinimidyl Ester fluorescently tagged with Alexa Fluor 647 and incubated at room temperature for 1 hour to ensure complete binding of the dye to free amines within the vECM. Fluorescently prelabeled biomaterials (500pL) were administered intravaginally as described above. Animals were euthanized 1, 2, or 3 days later (n=2 / time point), and vaginas were harvested and prepared for imaging and analyses.Histological Assessment of Epithelial Thickness

[0095] Five tissue sections spanning the length of the vagina were stained with hematoxylin and eosin and imaged with an Olympus VS200 slide scanning microscope at 20X magnification. Using QuPath v5 software, uniform gridlines were superimposed on each tissue cross-section, and 10-15 measurements of epithelial thickness were made where the epithelium intersected the grid to assure thorough sampling. The measurements were averaged for each section and again across all sections for each biological replicate.Histological Assessment of Epithelial Stem Cells

[0096] Tissue sections were incubated with DAPI nuclear stain, and antibodies against epithelial stem cell nuclear marker p63 (OriGene) and against a vaginal epithelium membrane marker filaggrin (Biorbyt), conjugated with an Alexa Fluor 647 or 488 secondary antibodies (Invitrogen), respectively. Tissue sections were imaged with an Olympus VS200 slide scanning microscope at 40X magnification. Using QuPath v5 software, uniform gridlines were superimposed on 5 sections per specimen, sampling throughout the length of the vagina, with five 250pm x 250pm grids of epithelium analyzed per each tissue section. The region of epitheliumwas traced using positive filaggrin signal and the QuPath cell counter was used to mark all nuclei within the grid to enable quantification of p63+cells within the epithelial region. The overall nucleated cell density (number of nuclei / epithelium region area); density of epithelial stem cells within the epithelium (number of p63+nuclei / epithelium region area), and proportion of epithelial stem cells (number of p63+nuclei per total number of nuclei within the epithelium region area). For all metrics, data were averaged for each section and across all sections per specimen.Histological Assessment of Vaginal Smooth Layer Muscle

[0097] Tissue sections were incubated with DAPI nuclear stain, and antibodies against alpha smooth muscle actin (Dako) and against a nuclear marker for proliferating cells Ki67 (Abeam), conjugated with an Alexa Fluor 568 or 647 secondary antibodies (Invitrogen), respectively. Tissue sections were imaged with an Olympus VS200 slide scanning microscope at 40X magnification. Using QuPath v5 software, uniform gridlines were superimposed on 5 sections per specimen, sampling throughout the length of the vagina, with 10-15 measurements analyzed per each tissue section to determine smooth muscle thickness. To quantitively assess smooth muscle proliferation, five 250pm x 250pm grids were analyzed per each tissue section. The region of smooth muscle within the grid was traced and QuPath cell counter was used to count the total number of nuclei and the number of Ki67+nuclei within the smooth muscle region. Data were averaged as described above.Histological Assessment of Macrophage Infiltrate and Phenotype Induced by the Study Treatments

[0098] Tissue sections were incubated with DAPI nuclear stain, and antibodies against a panmacrophage membrane marker CD68 (Abeam) and against a marker of M2 macrophages CD 163 (BioRad), conjugated with an Alexa Fluor 568 or 448 secondary antibodies (Invitrogen), respectively. Tissue sections were imaged with an Olympus VS200 slide scanning microscope at 40X magnification. Using QuPath v5 software, uniform gridlines were superimposed on 5 sections per specimen, sampling throughout the length of the vagina. To assess macrophage density, five 250pm x 250pm grids were analyzed per each tissue section. QuPath cell counter was used to count the total number of nuclei, and the number of CD68+and CD163+nuclei within each grid. Data were averaged as described above.

[0099] For the short-timepoint retention study, tissue sections were stained against DAPI, CD68, and CD163 as described and imaged with an Olympus VS200 slide scanning microscope.All tissue sections were inspected for presence of cells in the lumen, fluorescently prelabeled material in the lumen, and fluorescently prelabeled material in the fibromuscularis. For these three metrics, percentage of sections with positive identifications were compared among the groups analyzed. Finally, for all sections with fluorescently prelabeled material in the lumen or fibromuscularis, the tissue section was exported to ImageJ, after which a uniform threshold was applied to all sections. The positive area of fluorescent prelabeled material signal was then measured for each section and averaged across all sections per animal.Statistical Analysis

[0100] For all analyses, a one-way ANOVA followed by post-hoc pairwise comparisons with Tukey’s test was used to compare data between all groups. Significance was set to 0.05 with two- sided testing. Outliers were not excluded; the quantitative analyses were conducted by the investigators blinded to the group identity. Data, presented as mean±standard error of the mean were analyzed using GraphPad Prism vl0.2, San Diego, CA.Example 2. ResultsFabrication and Characterization ofvECM Hydrogel

[0101] Given the lack of existing published protocols for a decellularized vaginal tissue- derived ECM hydrogel, extensive optimization of a new decellularization protocol was required, informed by a previous successful workflow used to develop ECM hydrogels from cardiac muscle and skeletal muscle.23A summary of the decellularization conditions tested in iterative optimization batches is shown in Table 1. Notably, the decellularization conditions previously utilized for skeletal and cardiac muscle hydrogels (Table 1, Row A) did not result in successful gelation of the material. In subsequent iterative decellularization optimization batches, tissue processing, sodium dodecyl sulfate (SDS) concentration, decellularization duration, and water rinsing steps were varied, as shown in Table 1. Batches were evaluated for gelation ability, residual DNA, residual SDS, and sulfated glycosaminoglycan (sGAG) content. Residual DNA and sGAG content were at acceptable levels in all batches,23while residual SDS was acceptable in all conditions except the first batch (Table 1, Row A). Gelation was only observed in the batches that utilized 3 days of 0.1% SDS decellularization (Table 1, Row D, E).24

[0102] Ultimately, the vaginal decellularization protocol necessitated a much lower concentration of SDS for a shorter duration - 0.1% for 3 days - with a longer water rinsing period of 3 days to remove residual SDS, as compared to established cardiac and skeletal muscledecellul arization protocols. The final decellularization protocol is depicted in Figure 1 . To further evaluate this final protocol, hematoxylin and eosin staining was performed on tissue cross-sections taken throughout the decellularization process, and it was found that ECM structure was maintained while nuclear content was sufficiently removed from the tissue (Figure 2, Panels A- D).Table 1. Decellularization conditions tested in optimization batches

[0103] vECM and native porcine tissues with ECM targeted proteomics were next analyzed using quantitative concatemers (QConCAT) as previously described.25 26Proteins were identified and categorized according to ECM function, and relative intensities of each category were found for porcine vaginal tissue and decellularized vECM (Figure 2, Panel E). vECM shared overall similar distribution in proteomic content, with a larger proportion of fibrillar collagen and smaller proportion of crosslinking enzyme, which is expected from the decellularization process.

[0104] vECM was then prepared at two concentrations, 6 and 8 mg / mL, which has been shown in previous studies to be viable concentrations for cellular interactions.27While 6 mg / mL has been determined to be the optimal concentration for intramuscular injections in terms of injectability, spread, and degradation,27the intravaginal application is unique in that it is applied topically in the lumen, and gelation into a solid hydrogel was not anticipated given the constant animal motion. As such, the 8 mg / mL formulation was tested in anticipation that increased viscosity may improve retention and efficacy in this application. The complex viscosity (Figure 2, Panel F) of 6 and 8 mg / mL vECM was tested, as well as turbidity to evaluate self-assembly at 37°C (Figure 2, Panel G). The 8 mg / mL demonstrated higher viscosity than 6 mg / mL vECM, although at a level stillamenable to delivery via a syringe applicator, and both materials were shear thinning. 8 mg / mL vECM demonstrated faster self-assembly (see time to 50% OD, Figure 2H), and 8 mg / mL also had a higher equilibrium OD than 6 mg / mL. vECM Improves Epithelial Thickness and Morphology in Ovariectomized Rats

[0105] The efficacy of intravaginally administered vECM for restoration of vaginal epithelium in ovariectomized (OVX) rats was also assessed. The study design (Figure 3, Panel A) utilized rats which were two weeks post-ovariectomy, which is a well-established model of surgically induced menopause.28Animals were randomly assigned to receive daily topical intravaginal administration of 500 pL of either vECM at two concentrations (6 or 8 mg / mL), collagen, or saline. Collagen was chosen as a control given it is the predominant component of vECM.

[0106] The primary outcome of interest was epithelial thickness, as epithelial atrophy is a hallmark of GSM.28Representative tissue sections for healthy controls and OVX animals treated with either saline, collagen, 6 mg / mL vECM, or 8 mg / mL vECM are shown in Figure 3, Panels B- F. Treatment with the higher concentration 8 mg / mL vECM resulted in significantly greater epithelial thickness compared to saline, collagen, and 6 mg / mL vECM. Even though 8 mg / mL vECM did not fully return the epithelial thickness to healthy values, it induced regeneration of the morphologically similar multilayered stratified epithelium (Figure 3, Panel F). These results also indicate that vECM has a dose dependent effect on epithelium in menopausal rats.

[0107] Epithelial stem cells were assessed in vaginal tissue sections from healthy controls and OVX animals treated with either saline, collagen, or 8mg / mL vECM. Previous studies have demonstrated that hypoestrogenism leads to decreased proliferation and differentiation of vaginal epithelial stem cells and a consequent decrease in vaginal epithelial stratification.6It has been shown that in healthy controls, the p63+epithelial stem cells constitute a relatively small portion of the total epithelial cells (Figure 4, Panel A). Conversely, in OVX saline treated animals, the p63+stem cells occupy almost the entirety of the vaginal epithelium, with very little differentiated epithelium present (Figure 4, Panel B). Importantly, vECM [8 mg / mL] treatment led to restoration of a healthy-appearing vaginal epithelial phenotype with respect to nuclei density (number of total nuclei normalized by epithelial area, Figure 4, Panel E), p63+nuclei density (number of epithelial stem cell nuclei normalized by epithelial area, Figure 4, Panel F), and proportion of p63+nuclei (number of epithelial stem cell nuclei normalized by total number of nuclei, Figure 4, Panel G). These analyses demonstrate that vECM treated animals display morphologically similar vaginalepithelium to healthy premenopausal animals and suggest that vECM enables epithelial stem cells to proliferate and differentiate as they would in healthy animals. vCM Modulates Smooth Muscle Thickness and Proliferation

[0108] In addition to epithelial atrophy, smooth muscle atrophy is a notable consequence of the hypoestrogenic state.29Thus, immunohistochemical staining against alpha smooth muscle actin (uSMA) was conducted to assess the smooth muscle layer thickness in all treatment groups. Representative immunohistochemical staining for non-OVX healthy controls, saline, collagen, 6 mg / mL vECM, and 8 mg / mL vECM groups are shown in Figure 5, Panels A-E, and quantitative comparison of these groups is shown in Figure 5, Panel F. Topical 8 mg / mL vECM treatment resulted in significantly greater smooth muscle thickness compared to saline, collagen, and 6 mg / mL vECM groups, with no significant differences relative to the healthy controls.

[0109] Tissue sections were additionally stained against the nuclear proliferation marker Ki67 alongside aSMA to assess the amount of proliferating smooth muscle cells. Representative immunohistochemical staining for healthy, saline, collagen, and 8 mg / mL vECM groups are shown in Figure 6, Panels A-D, and quantitative comparison is shown in Figure 6, Panels E-F. vECM treatment significantly improved both the density of nuclei within the smooth muscle layer (Figure 6, Panel E) and the percentage of proliferating nuclei within the smooth muscle (Figure 6, Panel F) compared to saline and collagen treatment. vECM group was not significantly different from healthy controls in either measurement. These data suggest that topical vECM treatment impacted smooth muscle regeneration and phenotype.Material Retention, Distribution, and Macrophage Interactions

[0110] Given the dose-dependent response of vaginal epithelial and smooth muscle regeneration in this model, a short timepoint material retention study was conducted. A single 500 pL dose of saline or fluorescently pre-labeled collagen, 6 mg / mL vECM, or 8 mg / mL vECM was delivered topically via intravaginal administration. Vaginal specimens were isolated at 1, 2, or 3 days post-administration to assess material spread and retention throughout the vaginal tissues. Fluorescently pre-labeled material was observed in all material groups (Figure 7, Panels A-C, cyan); interestingly, material was not only observed in the vaginal lumen, where it was delivered, but also in the fibromuscular layer. Material retention throughout the vagina was quantified by averaging the area of material across all vaginal sections per animal (Figure 7, Panel D), but no significant differences in material retention were observed between groups at any timepoint. Giventhe observation of material in the fibromuscularis, the frequency of observation was quantified by the percentage of sections that contained material in the fibromuscularis (Figure 7, Panel E). This phenomenon was observed in all material groups, but only 8 mg / mL vECM had significant increases over collagen over the 3 days.[0U1] As both collagen and vECM materials are too large to passively bypass the vaginal epithelial layer, it was hypothesized that cellular trafficking may be aiding the transfer of materials from the vaginal lumen to fibromuscularis. Indeed, in all events where vECM or collagen was observed in subepithelial layers, it was co-localized with nuclei. To further investigate, tissue sections from all groups were stained against macrophages, as they are a cornerstone of the immune response to biomaterials. In particular, macrophages demonstrate unique response to the acellular ECM scaffolds30and facilitate the pro-regenerative activity of ECM biomaterials by activating endogenous tissue repair mechanisms.30,31Representative tissue sections from each group are shown in Figure 7, Panel F, in which fluorescently pre-labeled collagen and vECM are shown in cyan, nuclei are shown in blue (DAPI), and macrophage membrane markers are shown in red (CD68, pan-macrophage marker). In all groups, macrophages and other cells without macrophage membrane marker labeling were seen in both the vaginal lumen and fibromuscularis, colocalized with the administered materials.

[0112] Given the data suggested the interaction of vECM and collagen with macrophages, macrophage density in vaginal tissues was assessed at the end of treatment, utilizing tissues from the efficacy study previously discussed. Tissues were stained against CD68 (pan-macrophage marker) and CD 163 (M2 macrophage marker). Representative immunofluorescent images for healthy controls (Figure 8, Panel A) and OVX animals treated with saline (Figure 8, Panel B), collagen (Figure 8, Panel C), or 8 mg / mL vECM (Figure 8, Panel D) are shown. The 6 mg / mL vECM group was not assessed for macrophages as this group did not demonstrate improvement in either epithelial or smooth muscle phenotype. There was no significant difference among groups in nuclei density (Figure 8, Panel E), but the vECM-treated group demonstrated significantly greater proportion of macrophages overall (Figure 8, Panel F) and specifically M2 macrophages (Figure 8, Panel G) compared to saline and collagen treated groups. vECM-treated group was not significantly different from healthy controls in either metric. These data indicate that vECM modulates the immune response in vaginal tissues to a more healthy phenotype.REFERENCESE Bachmann, G. & Santen, R. J. Clinical manifestations and diagnosis of genitourinary syndrome of menopause (vulvovaginal atrophy), at <www.uptodate.com>2. Wysocki, S., Kingsberg, S. & Krychman, M. Management of Vaginal Atrophy: Implications from the REVIVE Survey. Clin Med Insights Reprod Health 8, CMRH.S14498 (2014).3. Hill, K. The demography of menopause. Maturitas 23, 113-127 (1996).4. Peacock Kimberly, K. K. Menopause. (StatPearls, 2020).5. Mac Bride, M. B., Rhodes, D. J. & Shuster, L. T. Vulvovaginal atrophy. Mayo Clin Proc 85, 87-94 Preprint at https: / / doi.org / 10.4065 / mcp.2009.0413 (2010)6. Ali, A., Syed, S. M., Jamaluddin, M. F. B., Colino-Sanguino, Y., Gallego-Ortega, D. & Tanwar, P. S. Cell Lineage Tracing Identifies Hormone-Regulated and Wnt-Responsive Vaginal Epithelial Stem Cells. Cell Rep 30, 1463-1477. e7 (2020).7. Goldstein, I., Dicks, B., Kim, N. N. & Hartzell, R. Multidisciplinary Overview of Vaginal Atrophy and Associated Genitourinary Symptoms in Postmenopausal Women. Sex Med 1, 44-53 Preprint at https: / / doi.org / 10.1002 / sm2.17 (2013)8. Boreham, M. K., Wai, C. Y., Miller, R. T., Schaffer, J. I. & Word, R. A. Morphometric analysis of smooth muscle in the anterior vaginal wall of women with pelvic organ prolapse. Am J Obstet Gynecol 187, 56-63 (2002).9. Ghosh, M., Rodriguez-Garcia, M. & Wira, C. R. The immune system in menopause: Pros and cons of hormone therapy. Journal of Steroid Biochemistry and Molecular Biology 142, 171-175 (2014).10. Tomlin, H. & Piccinini, A. M. A complex interplay between the extracellular matrix and the innate immune response to microbial pathogens. Immunology 155, 186-201 Preprint at https: / / doi.org / 10T 111 / imm.12972 (2018)11. Tyagi, T., Alarab, M., Leong, Y., Lye, S. & Shynlova, O. Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of postmenopausal women with severe pelvic organ prolapse. J Cell Mol Med 23, 2907-2919(2019). Bachmann, G., Santen, R. J., Barbieri, R. L. & Burstein, H. J. Treatment of genitourinary syndrome of menopause (vulvovaginal atrophy), at <www.uptodate.com> Reiter, S. Barriers to effective treatment of vaginal atrophy with local estrogen therapy. Int J Gen Med 6, 153 (2013). Szymanski, J. K., Slabuszewska-Jozwiak, A. & Jakiel, G. Vaginal Aging — What We Know and What We Do Not Know. International Journal of Environmental Research and Public Health 2021, Vol. 18, Page 4935 18, 4935 (2021). Naumova, I. & Castelo-Branco, C. Current treatment options for postmenopausal vaginal atrophy. Int J Womens Health 10, 387-395 Preprint at https: / / doi.org / 10.2147 / IJWH.S158913 (2018) Angelou, K., Grigoriadis, T., Diakosavvas, M., Zacharakis, D. & Athanasiou, S. The Genitourinary Syndrome of Menopause: An Overview of the Recent Data. Cureus 12, (2020). Alperin, M., Burnett, L., Lukacz, E. & Brubaker, L. The mysteries of menopause and urogynecologic health: Clinical and scientific gaps. Menopause 26, 103-111 (2019). Albalawi, N. S., Almohammadi, M. A. & Albalawi, A. R. Comparison of the Efficacy of Vaginal Hyaluronic Acid to Estrogen for the Treatment of Vaginal Atrophy in Postmenopausal Women: A Systematic Review. Cureus 15, e44191 (2023). You, S., Liu, S., Dong, X., Li, H., Zhu, Y. & Hu, L. Intravaginal Administration of Human Type III Collagen-Derived Biomaterial with High Cell-Adhesion Activity to Treat Vaginal Atrophy in Rats. ACS Biomater Sci Eng 6, 1977-1988 (2020). DeQuach, J. A., Lin, J. E., Cam, C., Hu, D., Salvatore, M. A., Sheikh, F. & Christman, K. L. Injectable skeletal muscle matrix hydrogel promotes neovascularization and muscle cell infiltration in a hindlimb ischemia model. Eur Cell Mater 23, 400-412 (2013). Spang, M. T. & Christman, K. L. Extracellular matrix hydrogel therapies: In vivo applications and development. Acta Biomater 68, 1-14 Preprint at https: / / doi.Org / 10.1016 / j.actbio.2017.12.019 (2018)Traverse, J. H., Henry, T. D., Dib, N., Patel, A. N., Pepine, C., Schaer, G. L , DeQuach, J. A., Kinsey, A. M., Chamberlin, P. & Christman, K. L. First-in-Man Study of a Cardiac Extracellular Matrix Hydrogel in Early and Late Myocardial Infarction Patients. JACC Basic Transl Sci 4, 659-669 (2019). Ungerleider, J. L., Johnson, T. D., Rao, N. & Christman, K. L. Fabrication and characterization of injectable hydrogels derived from decellularized skeletal and cardiac muscle. Methods 84, 53-59 (2015). Kasravi, M., Ahmadi, A., Babajani, A., Mazloomnejad, R., Hatamnejad, M. R., Shariatzadeh, S., Bahrami, S. & Niknejad, H. Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine. Biomaterials Research 2023 27:1 27, 1-24 (2023). Johnson, T. D., Hill, R. C., Dzieciatkowska, M., Nigam, V., Behfar, A., Christman, K. L. & Hansen, K. C. Quantification of decellularized human myocardial matrix: A comparison of six patients. Proteomics Clin Appl. 10, 75-83 (2016). Hill, R. C., Calle, E. A., Dzieciatkowska, M., Niklason, L. E. & Hansen, K. C. Quantification of extracellular matrix proteins from a rat lung scaffold to provide a molecular readout for tissue engineering. Mol Cell Proteomics 14, 961-973 (2015). Hernandez, M. J., Zelus, E. I., Spang, M. T., Braden, R. L. & Christman, K. L. Dose optimization of decellularized skeletal muscle extracellular matrix hydrogels for improving perfusion and subsequent validation in an aged hindlimb ischemia model. Biomater Sci 8, 3511-3521 (2020). Mao, M., Li, Y., Zhang, Y., Kang, J. & Zhu, L. Tissue Composition and Biomechanical Property Changes in the Vaginal Wall of Ovariectomized Young Rats. Biomed Res Int 2019, 8921284 (2019). Kim, N. N., Min, K., Pessina, M. A., Munarriz, R., Goldstein, I. & Traish, A. M. Effects of ovariectomy and steroid hormones on vaginal smooth muscle contractility. International Journal of Impotence Research 2004 16: 1 16, 43-50 (2004). Brown, B. N., Valentin, J. E., Stewart-Akers, A. M., McCabe, G. P. & Badylak, S. F. Macrophage phenotype and remodeling outcomes in response to biologic scaffolds withand without a cellular component. Biomaterials 30, 1482 (2009). Badylak, S. F., Valentin, J. E., Ravindra, A. K., McCabe, G. P. & Stewart-Akers, A. M. Macrophage phenotype as a determinant of biologic scaffold remodeling. Tissue Eng Part A 14, 1835-1842 (2008). Sueters, J., Xiao, F., Roovers, J.-P., Bouman, M.-B., Groenman, F., Maas, H., Huirne, J. & Smit, T. Creation of a decellularized vaginal matrix from healthy human vaginal tissue for potential vagina reconstruction: experimental studies. (2023). Hou, C., Zheng, J., Li, Z., Qi, X., Tian, Y., Zhang, M., Zhang, J. & Huang, X. Printing 3D vagina tissue analogues with vagina decellularized extracellular matrix bioink. Tnt J Biol Macromol 180, 177-186 (2021). Zheng, J., Zhang, X., Guo, K., Yan, L., Xu, X., Shi, W., Zhang, J., Du, Y., Zhang, M. & Huang, X. Enhancing vaginal reconstruction through 3D bioprinted scaffolds using a novel vECM-GelMA-SF bioink. Biofabrication 17, 015030 (2024). Ungerleider, J. L., Johnson, T. D., Rao, N. & Christman, K. L. Fabrication and Characterization of Injectable Hydrogels Derived from Decellularized Skeletal and Cardiac Muscle. Methods 84, 53 (2015). Syed, O , Walters, N. J., Day, R. M., Kim, H. W. & Knowles, J. C. Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering. Acta Biomater 10, 5043-5054 (2014). Ventura, R. D., Padalhin, A. R., Park, C. M. & Lee, B. T. Enhanced decellularization technique of porcine dermal ECM for tissue engineering applications. Materials Science and Engineering: C 104, 109841 (2019). Benini, V., Ruffolo, A. F., Casiraghi, A., Degliuomini, R. S., Frigerio, M., Braga, A., Serati, M., Torella, M., Candiani, M. & Salvatore, S. New Innovations for the Treatment of Vulvovaginal Atrophy: An Up-to-Date Review. Medicina (B Aires) 58, 770 (2022). Goldstein, I., Dicks, B., Kim, N. N. & Hartzell, R. Multidisciplinary Overview of Vaginal Atrophy and Associated Genitourinary Symptoms in Postmenopausal Women. Sex Med 1, 44-53 (2013).Tomczyk, K., Chmaj-Wierzchowska, K., Wszolek, K. & Wilczak, M. New Possibilities for Hormonal Vaginal Treatment in Menopausal Women. Journal of Clinical Medicine 2023, Vol. 12, Page 4740 12, 4740 (2023). Tylek, T., Wong, J., Vaughan, A. E. & Spiller, K. L. Biomaterial-mediated intracellular control of macrophages for cell therapy in pro-inflammatory and pro-fibrotic conditions. Biomaterials 308, 122545 (2024). Hong, J. Y., Seo, Y., Davaa, G., Kim, H. W., Kim, S. H. & Hyun, J. K. Decellularized brain matrix enhances macrophage polarization and functional improvements in rat spinal cord injury. Acta Biomater 101, 357-371 (2020). Savitri, C., Ha, S. S., Liao, E., Du, P. & Park, K. Extracellular matrices derived from different cell sources and their effect on macrophage behavior and wound healing. J Mater Chem B 8, 9744-9755 (2020). Tian, G., Jiang, S., Li, J., Wei, F., Li, X., Ding, Y., Yang, Z , Sun, Z., Zha, K., Wang, F. X., Huang, B., Peng, L., Wang, Q., Tian, Z., Yang, X., Wang, Z., Guo, Q., Guo, W. & Liu, S. Cell-free decellularized cartilage extracellular matrix scaffolds combined with interleukin 4 promote osteochondral repair through immunomodulatory macrophages: In vitro and in vivo preclinical study. Acta Biomater 127, 131-145 (2021). Liu, C., Wang, K., Liu, W., Zhang, J., Fan, Y. & Sun, Y. ALOX15+ M2 macrophages contribute to epithelial remodeling in eosinophilic chronic rhinosinusitis with nasal polyps. Journal of Allergy and Clinical Immunology 154, 592-608 (2024). Raveenthiraraj, S., Awanis, G., Chieppa, M., O’Connell, A. E. & Sobolewski, A. Ml and M2 Macrophages Differentially Regulate Colonic Crypt Renewal. Inflamm Bowel Dis 30, 1138-1150 (2024). Hung, L. Y., Sen, D., Oniskey, T. K., Katzen, J., Cohen, N. A., Vaughan, A. E., Nieves, W., Urisman, A., Beers, M. F., Krummel, M. F. & Herbert, D. B. R. Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism. Mucosal Immunology 2018 12:1 12, 64-76 (2018). Yu, Y., Yue, Z., Xu, M., Zhang, M., Shen, X., Ma, Z., Li, J. & Xie, X. Macrophages playa key role in tissue repair and regeneration. PeerJ 10, el4053 (2022). Li, W., Xu, Z , Zou, B., Yang, D., Lu, Y., Zhang, X , Zhang, C , Li, Y. & Zhu, C. Macrophage regulation in vascularization upon regeneration and repair of tissue injury and engineered organ transplantation. Fundamental Research (2024). doi:10.1016 / J.FMRE.2023.12.015 Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nature Methods 2009 6:5 6, 359-362 (2009).

Claims

CLAIMSWhat is claimed is:

1. A biocompatible material comprising decellularized extracellular matrix derived from vaginal tissue (vECM).

2. The biocompatible material of claim 1, wherein said vECM is in a liquid or gel form.

3. The biocompatible material of claim 1, wherein said vECM is in a concentration of greater than about 6 mg to about 20 mg of vECM per mb of the biocompatible material.

4. The biocompatible material of claim 1, wherein said vECM is in a concentration of about 8 mg of vECM per mL of the of the biocompatible material.

5. The biocompatible material of claim 1, wherein said vaginal tissue is selected from the group consisting of human vaginas, porcine vaginas, bovine vaginas, goat vaginas, mouse vaginas, rat vaginas, rabbit vaginas, and chicken vaginas.

6. A method of treating vaginal morbidities, including vaginal atrophy (thinning of the epithelial layer, loss of vaginal rugae, loss of smooth muscle layer), insufficient vaginal lubrication, vaginal burning, vaginal pruritis, vaginal pain, dyspareunia, fibrosis, scarring / adhesions, stenosis, shortening, pallor, firmness, telangiectasias, fragility, exposure of the implanted synthetic grafts, granulation tissue due to reaction to the implanted synthetic grafts or sutures, bleeding, vaginal epithelial abnormalities, ulcerations, and alterations in vaginal mechanical properties, such as decreased elasticity, increased stiffness, comprising administering to a person in need thereof an effective amount of a composition comprising decellularized extracellular matrix (ECM).

7. The method of claim 6, wherein the composition comprises a concentration of greater than about 6 mg to about 20 mg of decellularized extracellular matrix derived from vaginal tissue (vECM) per mL of the composition.

8. The method of claim 6, wherein the composition comprises a concentration of about 8 mg of decellularized extracellular matrix derived from vaginal tissue (vECM) per mL of the composition.

9. The method of claim 6, wherein the decellularized extracellular matrix is derived from vaginal tissue, striated muscle or smooth muscle, small intestine submucosa, bladder, uterus, heart, liver, lung, or blood vessels.

10. The method of claim 6, wherein said composition is coated on an implant or a molded insert.

11. The method of claim 6, wherein said composition is delivered as a liquid.

12. The method of claim 6, wherein said composition transitions to a gel form after delivery.

13. The method of claim 6, wherein said composition degrades within one day to three months following delivery.

14. The method of claim 6, wherein the composition is administered more than once.

15. The method of claim 6, wherein the delivery is transvaginal.

16. The method of claim 6, wherein the delivery is intravaginal.

17. The method of claim 6, wherein said composition is a wash, a liquid, a particulate, or a hydrogel.

18. The method of claim 6, wherein said composition is injected through a needle of less than 30 gauge into vaginal tissue.

19. The method of claim 6, wherein the composition further comprises an additional component selected from the group consisting of a protein, peptide, polypeptide, exogenously added cell, autologous cell, cellular growth factor, nutrient, synthetic polymer, survival promoting additive, proteoglycan, glycosaminoglycan, therapeutic agent, and a combination thereof.

20. A method of formulating a vaginal extracellular matrix (vECM) composition, the method comprising: isolating a vaginal tissue sample; decellularizing the tissue sample; lyophilizing the tissue sample; milling the tissue sample; and enzymatically digesting the tissue sample; wherein the vECM composition is effective to treat a vaginal morbidity in a subject.

21. The method of claim 20, wherein the vaginal tissue sample comprises vaginal tissue selected from the group consisting of human vaginas, porcine vaginas, bovine vaginas, goat vaginas, mouse vaginas, rat vaginas, rabbit vaginas, and chicken vaginas.

22. The method of claim 20, wherein the vaginal morbidity is selected from vaginal atrophy (thinning of the epithelial layer, loss of vaginal rugae, loss of smooth muscle layer), insufficient vaginal lubrication, fibrosis, scarring / adhesions, stenosis, shortening, pallor, firmness, telangiectasias, fragility, exposure of the implanted synthetic grafts, granulation tissue due to reaction to the implanted synthetic grafts or sutures, bleeding, ulcerations, and alterations in vaginal mechanical properties, such as decreased elasticity, increased stiffness.

23. The method of claim 20, wherein the vECM composition comprises a concentration of greater than about 6 mg to about 20 mg of vECM per mb of the vECM composition.

24. The method of claim 20, wherein the vECM composition comprises a concentration of about 8 mg of vECM per m of the vECM composition.

25. The method of claim 20, wherein the decellularization step comprises about 3 days of 0.1% SDS decellularization.

26. The method of claim 25, wherein the decellularization step further comprises about 3 days of rinsing to remove SDS.

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