Improved methods for decellularization of extracellular matrix (ECM) and preparation of decellularized ECM gels and uses thereof

Abstract

The present disclosure describes a novel method for decellularizing amphibian extracellular matrix to obtain isolated ECM that is substantially free of cellular and nuclear debris. The method includes washing the amphibian ECM in a solution containing one or more detergents, incubating the ECM with one or more proteases, and incubating the ECM with one or more nucleases. The present disclosure also describes various methods of using the isolated ECM.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 267,867, filed February 11, 2022, which is incorporated by reference herein in its entirety.

[0002] The present disclosure describes novel methods for decellularizing extracellular matrix and compositions comprising decellularized extracellular matrix for a variety of uses. [Background technology]

[0003] Skin, the largest organ of the mammalian body, is the outer covering of the body that serves three main functions: protection from microorganisms and excess water loss, control of body temperature, and the perception of touch, heat, and cold. Mammalian skin has two main layers, the epidermis and the dermis. The epidermis is the outermost layer of skin that prevents invading microorganisms and retains moisture in the body; it is a stratified squamous epithelium composed of keratinocytes. The dermis is the layer of skin just below the epidermis, which serves as a site for the skin's appendages and gives the skin elasticity through an extracellular matrix composed of collagen fibers, elastic fibers, hyaluronan, and proteoglycans. The dermis and epidermis are separated by a thin sheet of fibers called the basement membrane, which controls the flow of cells and molecules such as cytokines and growth factors between the dermis and epidermis during the processes of remodeling, repair, and renewal. Beneath the dermis is the subcutaneous tissue, which is composed of loose connective tissue such as fat and elastin. The main cells of the subcutaneous tissue include fibroblasts, macrophages, and adipose tissue.

[0004] Injury or disease results in disturbances in the morphology and function of an organ or tissue, such as the skin. Remodeling and repair of an organ or tissue after injury is a complex wound healing process that involves interactions between cells, growth factors, and the extracellular matrix (ECM). The process in adult mammals includes well-known stages: homeostasis, inflammation, proliferation, maturation, and remodeling. During homeostasis, coagulation occurs to stop bleeding. Inflammation involves the recruitment of white blood cells, antibodies, nutrients, and enzymes to the affected area to accelerate wound healing. During proliferation, new healthy granulation tissue, including new connective tissue and blood vessels, replaces the wound. Maturation and remodeling occur after the wound is closed and include repair of the dermal tissues to improve their tensile strength.

[0005] In contrast to repair processes, where the goal is to re-establish function regardless of the exact location of the injured tissue, regeneration is the replacement of injured tissue with an exact replica such that both morphology and functionality are fully restored. As an example, uninjured skin undergoes complete regeneration with a constant replacement of new cells. However, injured adult mammalian skin does not completely regenerate and heals with a scar.

[0006] In contrast to mammals, urodeles regenerate their skin structures, including the dermis and secretory glands, after deep skin injury without scar formation. It has been reported that the extracellular matrix (ECM) of urodeles may have desirable properties important for wound healing, and therefore, the ECM may be an ideal source of biomaterials for wound healing, xenografts, and other skin conditions. Thus, urodeles ECM may provide a model for tissue repair and regeneration in mammals, as well as a starting material for compositions for treating mammalian tissues, including the skin.

[0007] The ECM of urodele contains natural cells and genetic material to maintain structural and biochemical functions and properties. However, the natural cells and genetic material of urodele ECM may induce undesirable effects and immune responses in subjects. Decellularization is a process in which the natural cells and genetic material are removed from tissue without affecting the structural and functional integrity of the tissue. The decellularized tissue can then be used in subjects. However, there is a need to develop improved methods for obtaining decellularized urodele ECM that can be effectively used in subjects, especially mammalian subjects. Summary of the Invention [Means for solving the problem]

[0008] This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0009] The present disclosure provides new and improved methods for decellularizing amphibian biological samples. In embodiments, the biological samples contain ECM and are taken from juvenile urodela (e.g., Ambystoma hybrids) or larval Apoda (frog / tadpole) species.

[0010] The method includes washing the sample with one or more detergents, and incubating the sample with one or more proteases and one or more nucleases. In an embodiment, the method includes washing the sample with one or more detergent solutions, treating the sample with one or more proteases, and treating the protease-treated sample with one or more nucleases to obtain isolated ECM that can be used in vitro and / or in vivo. The one or more detergent solutions include one or more non-ionic detergents, such as Triton X-100™ reduced, and / or anionic detergents, such as N-lauryl sarcosine (NLS). The one or more proteases include amino endopeptidases, such as Dispase II. The one or more nucleases include DNA / RNA endonucleases, such as Benzonase® nuclease. After decellularization, the isolated ECM sample can be micronized and dehydrated for storage.

[0011] In embodiments, a method for decellularizing a sample of ECM includes washing the sample with a detergent solution containing an NLS; treating the washed sample with one or more proteases, such as dispase II; treating the protease-treated sample with a DNA / RNA endonuclease, such as benzonuclease; and dehydrating the sample under mild conditions without particulation.

[0012] The isolated ECM sample can be prepared into different forms, for example, gelatinized ECM in the form of gelatin.

[0013] The present disclosure also describes an isolated ECM and a composition comprising the isolated ECM. The isolated ECM has more than 70% of the cells in the ECM removed, but the structural and / or functional architecture of the ECM remains intact. The isolated ECM contains less than 20 ng / mg of dry ECM (or less than 10 ng / μl of DNA extract). The length of the residual DNA fragments in the isolated ECM is less than 300 base pairs (bp).

[0014] The isolated ECM and compositions can be used for organ and tissue regeneration, as implants, and to treat skin conditions. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with TritonX-100 shown. [Figure 1B] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with TritonX-100 shown. [Figure 1C] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with TritonX-100 shown. [Figure 1D] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with TritonX-100 shown. [Figure 1E] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with NLS shown. [Figure 1F] FIG. 1 shows a slide of isolated ECM stained with hematoxylin and eosin and imaged by light microscopy, with a sample washed with NLS shown. [Figure 2A] FIG. 1 shows the amount of DNA in isolated ECM. Average DNA quantification per sample is shown. [Figure 2B] FIG. 2B shows the amount of DNA in isolated ECM, showing the percentage of DNA converted from the concentrations shown in FIG. 2A. [Figure 3A]1% agarose gel with DNA ladder showing residual DNA from naive tissue samples and isolated ECM samples (Triton X-100 (T1 or T2) samples or NLS samples (NLS): T1_BLC, T_C, T2_BLC, T2_C, NLS_BLC, and NLS_C from the bottom left corner (BLC) or center (C) of the isolated ECM samples). [Figure 3B] FIG. 1 shows a 1% agarose gel with a DNA ladder showing residual DNA from isolated ECM samples in triplicate (3×, 3 lanes). [Figure 4A] FIG. 1 shows the average particle length with error bars for the freeze-milled samples. The average lengths for T1 and NLS are 28.36 μm±13.19 and 30.05 μm±34.70, respectively. [Figure 4B] Figure 14 shows the average particle length with error bars for the freeze-milled samples. The average areas for T1 and NLS are 494.06 μm2 ± 293.2 and 631.5 μm2 ± 550.91, respectively. [Figure 5A] FIG. 1 shows the results of an adhesion assay. The graph compares the optical density (OD) of T1, T2, and NLS using 32 μl, 64 μl, and 128 μl protein concentrations for fibroblasts. A control group was included to assess normal adhesion without ECM protein (bare wells). [Figure 5B] FIG. 1 shows the results of an adhesion assay. The graph compares the optical density (OD) of T1, T2, and NLS using 32 μl, 64 μl, and 128 μl protein concentrations for HeCaT cells. A control group was included to assess normal adhesion without ECM protein (bare wells). [Figure 6A] Figure 1 shows the results of a proliferation assay: OD measurements at 570 nm wavelength for all treatment modalities at 24, 48 and 72 hours for fibroblasts. [Figure 6B] Figure 1 shows the results of a proliferation assay: OD measurements at 570 nm wavelength for all treatment modalities at 24, 48 and 72 hours for HeCaT cells. [Figure 7A] Figure 1 shows the results of the migration assay. Intensity measurements to evaluate the migration of fibroblasts under different conditions (T1, T2, NLS, and control). All wells were coated with the same concentration as in the proliferation assay, as determined by the attachment assay. [Figure 7B] Figure 1 shows the results of the migration assay. Intensity measurements to evaluate the migration of HeCaT cells under different conditions (T1, T2, NLS, and control). All wells were coated with the same concentration as in the proliferation assay, as determined by the attachment assay. [Figure 8] FIG. 1 shows the recorded mass shown on a line graph, with an observable decrease in mass as the decellularization process progresses. The spike in mass can be explained as the ability of the ECM to retain a greater amount of water as it becomes decellularized. [Figure 9A] FIG. 1 shows histology slide images of naive tissue. [Figure 9B] FIG. 1 shows the histology slide image in the top left corner (TLC). [Figure 9C] FIG. 1 shows the center histology slide image. [Figure 10]

[0033] Figure 1 shows the average DNA quantification per sample. NLS dried samples yielded approximately 17 ng of DNA per milligram of dried axolotl tissue. [Figure 11] FIG. 1 shows images of 1% agarose gel for DNA ladder, naive tissue DNA sample, top left corner (TLC), center, and dried (NLS dry 19 mg) acquired on an Azure Biosystems c150. [Figure 12] FIG. 1 shows the optical density (OD) for protein concentrations of 32 μL, 64 μL, and 128 μL for NLS with gentle dehydration. A control group was included to assess normal adhesion without ECM protein, i.e., bare wells. [Figure 13]FIG. 1 shows OD measurements at 570 nm wavelength for fibroblasts and HeCaT cells at 24, 48, and 72 hours for all treatment modalities. [Figure 14] Figure 1 shows the results of a migration assay. Intensity measurements were performed to assess the migration of fibroblasts and HeCaT cells on NLS-treated samples with mild dehydration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," "the," and similar referents are to be construed to cover both the singular and the plural, unless otherwise indicated in the specification or clearly contradicted by context.

[0017] The term "antigen" refers to a molecule that is toxic or foreign to a subject and induces an immune response in the form of the production of antibodies against that molecule. The isolated ECM described herein can have reduced antigenicity compared to native ECM so that it can be used in a subject.

[0018] The term "biological activity" refers to a biological effect. Biologically active or biologically active substances include substances that have a biological function.

[0019] The term "biocompatible" refers to a product and its normal degradation products, in vitro, ex vivo, or in vivo, that is substantially non-toxic and non-carcinogenic to a cell, tissue, organ, organism, or subject, within useful, practical, and / or acceptable tolerances. The term "cytocompatible" refers to a product that is capable of sustaining the viability and growth of a population of cells.

[0020] The term "biomaterial" refers to a material suitable for in vitro, ex vivo, or in vivo use. As an example of an in vivo use, the biomaterial is suitable for administration to a subject in need thereof. The material may be synthetic or natural. The decellularized ECM (isolated ECM) described herein is an example of a biomaterial.

[0021] The term "carrier" or "excipient" refers to a substance added to a composition that does not affect the active compound in the composition. A carrier can be a diluent. An excipient can be a substance added to a composition to facilitate administration of the composition.

[0022] The term "cosmetics" refers to products (other than pure soaps) intended to be applied to the human body to cleanse, beautify, enhance attractiveness, or change appearance. Examples of cosmetic benefits can include improving the appearance of skin, such as improving the appearance of wrinkles and fine lines, removing oil and excess sebum, diluting the appearance of skin blemishes, cleansing and conditioning the skin, evening skin tone, firming the skin, soothing inflammation, and refreshing and cooling the skin.

[0023] The terms "drug," "medicine," or "therapeutic substance" refer to any article (other than food) intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, and any article intended to affect the structure or any function of the body of humans or other animals.

[0024] The terms "derive," "derived," or "derives" refer to products obtained by any useful method from any referenced source. For example, amphibian-derived extracellular matrix (ECM) refers to ECM obtained from members of the amphibian family.

[0025] The term "exogenous" refers to a product that originates outside of the organism, tissue, cell, organ, or subject. In contrast, the term "endogenous" refers to a product that originates from the organism, cell, tissue, organ, or subject.

[0026] The term "extracellular matrix" or "ECM" refers to a natural scaffold with a three-dimensional structure that contains biomolecules and minerals that provide biochemical support to surrounding cells. ECM includes structural and non-structural biomolecules such as collagen, elastin, laminin, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. ECM can be obtained from a variety of sources of tissue, including dermal and non-dermal tissues.

[0027] The term "decellularized extracellular matrix" or "decellularized ECM" refers to ECM prepared by removing cells and / or deactivating cells from ECM found in multicellular organisms, e.g., amphibians or mammals. Decellularized ECM is substantially free of cellular components, including intact cells, lysed cells, and cellular and nuclear debris, and therefore, decellularized ECM exhibits reduced immunogenicity and thus can be administered to a subject, e.g., a mammalian subject, as a non-toxic xenograft or biomaterial. Decellularized ECM that is "substantially free of immunogenic components" refers to ECM in which immunogenic components are present at levels that are not sufficient to induce an adverse immune response in a subject.

[0028] The term "isolated" refers to something that has been separated or removed from its natural surroundings such that it is substantially free of components with which it is associated in its naturally occurring state. For example, a cell or protein may be isolated from its naturally occurring state. The term "isolated ECM" refers to decellularized ECM.

[0029] The term "immunogenic" refers to relating to or producing an immune response. The term "immunogenicity" refers to the ability of a foreign substance, such as an antigen, to elicit an immune response in a subject. The isolated ECM described herein can have reduced immunogenicity compared to native ECM, and therefore, it can be used as a biomaterial in a subject.

[0030] The terms "native ECM" or "naive ECM" refer to naturally occurring amphibian ECM (or the corresponding ECM sample) that has not been decellularized.

[0031] The term "non-toxic" refers to a product that causes little adverse reaction or substantial harm to cells and tissues in vitro or ex vivo and / or causes no substantial harmful or undesirable reaction or substantial harm to cells and tissues in the body (in vivo).

[0032] The term "prevent" or "prevention" refers to the prevention of the occurrence, recurrence, or spread of a condition or one or more symptoms of a condition. By way of example, the condition may be a skin condition. The term includes administration of a product described herein prior to the occurrence of a symptom, particularly to a subject at risk of developing a condition such as a skin condition. The term includes the inhibition or reduction of one or more symptoms associated with a skin condition. The term "prevention" may be used interchangeably with the term "prophylactic treatment."

[0033] The term "retaining structural and functional integrity" as used in reference to ECM refers to retaining sufficient structure and function to permit and support use of the matrix as a substrate for cell growth in vivo, ex vivo, or in vitro. For example, isolated ECM retains the structural and functional properties of naturally occurring ECM that permit its use as a biomaterial.

[0034] The terms "scaffold" and "bioscaffold" are used interchangeably to refer to a substrate on which cells can grow in vitro, ex vivo, and / or in vivo. A scaffold or bioscaffold is an example of a biomaterial.

[0035] The term "skin condition" includes skin conditions that require therapeutic "medicinal" treatment, such as diseases, defects, and injuries including wounds.

[0036] The term "cosmetic skin condition" includes skin conditions related to skin tone, clarity, radiance, lightness, and / or hydration.

[0037] The term "subject" refers to an animal, e.g., a mammal. Examples of mammals include humans, dogs, cats, horses, cows, goats, sheep, pigs, or non-human primates. Subjects in need of treatment or in need thereof include subjects having a disease or condition that requires treatment. Subjects in need thereof also include subjects in need of treatment and / or prevention of a skin condition.

[0038] The term "therapeutically effective amount" refers to an amount of a product or composition, e.g., a formulation, that provides a therapeutic benefit in the treatment, prevention, or management of a condition or disease, such as a skin disorder, injury to the skin, or a wound. The term "therapeutically effective amount" also includes an amount of a compound that, when administered, is sufficient to prevent the occurrence of, or to alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.

[0039] The term "treatment" or "treating" in the context of administering a product, such as a biomaterial, to a subject refers to administering the product to achieve a desired clinical / medical endpoint, e.g., to alleviate the symptoms of a disease or condition. Examples of such desired endpoints associated with skin diseases or conditions include wound healing, tissue closure, tissue plumping, preventing tissue adhesions, providing structural support to tissue, providing a protective barrier, and / or correcting defects. Administering a product also includes applying the product onto a subject.

[0040] The term "heterologous" refers to a product derived or originating from a member of another species.

[0041] The term "amphibians" refers to cold-blooded vertebrates including frogs, toads, newts, and salamanders belonging to the orders Urodela and Apoda. They have an aquatic, gill-breathing larval stage followed by a terrestrial, lung-breathing adult stage. Amphibians as described herein are juvenile or pycnomorphic amphibians. Juvenile amphibians include juvenile frogs such as froglets, tadpoles, or juvenile Apoda (frog / tadpole) species in the larval stage.

[0042] The term "salamander" refers to a group of amphibians characterized by a lizard-like appearance and the possession of a tail throughout life. There are ten families of salamanders, including Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), and Amphiutidae (siberian salamanders). salamanders), Proteidae (mudpuppies and olms), Rhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The ten families of salamanders are together classified in the order Urodela (or Caudata). The term "Urodele" refers to salamanders of the order Urodela in the class Amphibians. Salamanders can belong to the orders Urodela and Apoda.

[0043] Caudata begin life as aquatic animals during the larval stage, and some undergo metamorphosis from a gilled juvenile form to an adult, terrestrial, lunged, air-breathing form. During metamorphosis, the physical characteristics of the urodel change in preparation for life on land. These changes include the resorption of the tail fin, thickening of the skin, development of skin glands, and the resorption of gills. Sexual maturity also occurs during this period in most urodeles. However, some families of urodeles are "paedomorphic", meaning that individuals of such families retain their juvenile aquatic form throughout life, even after reaching sexual maturity. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of paedomorphic salamanders. Instead of becoming a terrestrial amphibian, adult axolotls remain aquatic and gilled, however, under certain circumstances, the axolotl undergoes metamorphosis and transforms into a terrestrial form.

[0044] Axolotls have the ability to completely regenerate lost or damaged body parts, including organs, limbs, and parts of the central nervous system, throughout their entire lifespan. Aquatic axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scarless processes. Similarly, metamorphosing terrestrial axolotls, which retain some larval skin characteristics, also exhibit scarless wound healing, but at a slower rate than their aquatic pre-metamorphosing counterparts. The axolotl wound healing process resembles the scarless healing process of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than the corresponding events occur in postnatal mammals. Furthermore, similar to the human amniotic membrane (AM), the axolotl ECM is rich in growth factors that favor wound healing.

[0045] The ECM is a three-dimensional network of extracellular polymers and minerals, including collagen, enzymes, glycoproteins, and hydroxyapatite, that provide structural and biochemical support to surrounding cells. The ECM may include a combination of fibrous and network collagens. Examples of various types of collagen include one or more of collagen types I, II, III, IV, V, and VI. The ECM may also include elastin and / or elastic fibers. The ECM may also include laminin, fibronectin, hyaluronan, chondroitin sulfate, or both, and / or one or more proteoglycans, glycoproteins, glycosaminoglycans, or any combination thereof. The components and structure of the ECM play an important role in the healing process, as the components of the ECM generate a scaffold that provides the structural architecture of the matrix required for the healing process. Furthermore, ECM components are involved in stimulating cell adhesion and migration during the healing process, as well as mediating interactions between cells, and between cells and the matrix, or between EMC proteins, during the healing process. Furthermore, ECM components also act as reservoirs and modulators of the action of cytokines and growth factors that control wound repair activity. However, ECM contains cells and genetic material that need to be removed before it can be used as a biomaterial, as these may induce harmful immune responses.

[0046] Decellularization of ECM is the removal of cells and cellular components from the ECM of a biological sample, such as a tissue, while retaining the ECM proteins and native ECM architecture or structure for effective use as a biomaterial. The present disclosure describes novel methods for decellularizing amphibian ECM, such as urodele ECM, for use in preventing and / or treating and curing a variety of conditions, including skin conditions. In contrast to known methods of decellularizing ECM, which may involve the use of crosslinking agents and other harsh agents, the methods described herein provide decellularized amphibian ECM that uses milder agents but contains fewer cells and cell debris, thus allowing the decellularized ECM to be used in vitro, ex vivo, and / or in vivo without causing undesirable adverse effects.

[0047] A method of decellularizing amphibian ECM includes harvesting or obtaining an amphibian sample, washing the sample, and incubating the sample with one or more proteases and one or more nucleases. In embodiments, a method of decellularizing amphibian ECM includes harvesting an amphibian sample, washing the sample, incubating the sample with one or more proteases, washing the protease-treated sample, and incubating the washed protease-treated sample with one or more nucleases to obtain isolated ECM that can be used as a biomaterial in vitro, ex vivo, and / or in vivo.

[0048] Amphibian samples include any biological sample from an amphibian, particularly a juvenile amphibian such as a juvenile frog or a paedomorphic mature urodele, that contains ECM and can be decellularized and used. Biological samples can include any amphibian tissue, such as connective tissue, adipose tissue, bone, plasma, skin, cartilage, tendon, dura mater, and fascia. Other tissues include dermis, basement membrane, and epithelial tissue, such as basement membrane or epithelial tissue lining body cavities, such as the parietal mesothelial tissue of the thoracic cavity, abdominal cavity, and pericardium. In an embodiment, the biological sample is a tissue from a juvenile amphibian, such as a juvenile frog or a paedomorphic mature urodele. The biological sample can be a fresh sample or a frozen sample that has been thawed. The sample can be from any species of urodele, including axolotls. In an embodiment, the sample is from axolotl skin.

[0049] Amphibian samples are obtained or harvested from juvenile amphibians, such as juvenile mature urodela or juvenile frogs. Samples are surgically removed from juvenile amphibian tissue. Samples can be full thickness explants of various shapes and sizes. Samples can be 5 x 5 centimeters (cm) or larger in size. Samples are cleaned and prepared for decellularization.

[0050] The decellularization methods described herein include briefly rinsing the sample with one or more solutions including water or a buffer solution, and washing with a detergent solution with agitation prior to protease treatment. Examples of buffer solutions include phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), Tris-buffered saline (TBS), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Detergents include non-denaturing non-ionic and anionic detergents that are gentle on the ECM and easy to use (e.g., can be used at room temperature without increasing or decreasing temperature or pressure). Non-ionic detergents such as Triton™ X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) and Triton™ X-100 reduced (polyoxyethylene(10) isooctylcyclohexyl ether) and secondary alcohol ethoxylates, e.g., TERGITOL™ (secondary alcohol ethoxylates) such as TERGITOL™ 15-2-9 (polyglycol ether surfactant) and TERGITOL™ TMN-10 (trimethylnonyl polyethylene glycol, polyethylene glycol trimethylnonyl ether), and anionic detergents such as N-lauryl sarcosine (NLS), potassium lauryl sarcosine, cholate, deoxycholate, sodium dodecyl sulfate (SDS), have been found to be useful mild detergents for decellularization of amphibian biological samples. Other non-ionic surfactants include polyoxyethylene sorbitan fatty acid esters or Tweens such as Tween 20, 40, 60, or 80.

[0051] Samples may be first rinsed at room temperature with water or a buffer solution at room temperature for 3-10 minutes, followed by a detergent wash at 37° C. with agitation. Exemplary washing conditions for detergent include: a 5×5 cm amphibian sample is washed twice with 20-30 ml of detergent in a buffer such as DPBS at a concentration of 0.1%-1.0% v / v, at 37° C. with agitation at 90-120 revolutions per minute (RPM), for 3-12 hours each time. In an embodiment, the sample is briefly washed in DPBS for 5 minutes at room temperature, followed by a wash with Triton, e.g., Triton X-100 or Triton X-100 reduced, or NLS, at 37° C. with agitation at 100 RPM for 6 hours, and then rinsed with a buffer solution such as PBS or DPBS at room temperature for 5 minutes. In an embodiment, the detergent solution comprises an anionic detergent such as NLS.

[0052] Samples can also be washed with a non-detergent solution containing latrunculin (Lat) or cytochalasins instead of detergent solutions. Lat is a cytochalasin that is an actin depolymerizing agent. They bind actin monomers near the nucleotide binding groove and prevent them from polymerizing. Examples of Lat include Lat A and Lat B. Examples of cytochalasins include cytochalasins A, B, C, D, E, F, H, and J. Lat has a potency in the nanomolar range. An exemplary non-detergent method using Lat for a 5x5 cm tissue sample is as follows: Incubate sample in 100 ml of 50 nanomolar (nM) Lat, e.g., Lat B, in high glucose DMEM for 2 hours (hr) at 37°C, wash with 100 ml double distilled water for 15 minutes (min), incubate with 100 ml 0.6 M potassium chloride for 90 minutes, incubate in 100 ml 1.0 molar (M) potassium iodide for 90 minutes, wash with 200 ml distilled water overnight, incubate with potassium chloride, incubate with potassium iodide, wash with distilled water overnight. Washes and incubations in high ionic solutions are performed at room temperature.

[0053] The washed amphibian sample is then incubated with one or more proteases at 37° C. for about 1 to 2 hours to remove the epidermis and disrupt cellular attachment to the dermal matrix. The concentration of the protease is at least 1 unit of enzyme per ml of solution, or 2 to 10 units / ml of solution, 2 to 7 units / ml of solution, 2 to 5 units / ml of solution, or 2 to 4 units / ml of solution. The one or more proteases for treating the amphibian sample include aminoendopeptidases, which are relatively mild and can dissociate sufficiently at physiological temperature and pH to maintain cell membrane integrity, thereby making the method easier to carry out. Examples of aminoendopeptidases include dispase, such as dispase I and dispase II, trypsin, papain, and collagenase. In an embodiment, the protease for treating the sample is dispase II. Dispase II has been found to have a mild proteolytic effect that helps maintain cell membrane integrity and is easy to use because it can be used at physiological temperature and pH. In an embodiment, a 5×5 cm tissue sample is incubated with 2 units of Dispase II per ml of DPBS at 37° C. for 90 minutes. In an embodiment, the Dispase is Dispase II (CAS number 42613-33-2).

[0054] The protease-treated sample is then rinsed briefly with one or more solutions, such as water or a buffer solution, at room temperature, hi an embodiment, the protease-treated sample is rinsed for 5 minutes with a buffered saline solution, such as PBS or DPBS.

[0055] After a brief rinse with PBS or DPBS, the protease-treated sample is then incubated with one or more nucleases for 3-12 hours at 37° C. with agitation at 90-120 RPM. The concentration of the nuclease is at least 30 units / ml of solution, or 40-100 units / ml, 40-75 units / ml, 40-60 units / ml, or 40-50 units / ml of solution. In this step, a non-specific DNA / RNA endonuclease is used to remove nucleic acids from the sample. Examples of non-specific endonucleases include benzonuclease and turbonuclease. Other nucleases include recombinant DNA nuclease, pulmozyme, and combinations of deoxyribonuclease and ribonuclease. In an embodiment, the washed protease treated sample is treated with 40 units / ml of benzonuclease for 6 hours. After nuclease treatment, the sample is rinsed briefly with a buffer solution such as PBS or DPBS at 37° C., then washed with a buffer solution such as PBS or DPBS for 24 hours at 37° C. with shaking at 100 RPM. In an embodiment, the nuclease is benzonase (CAS number 9025-65-4).

[0056] In an embodiment, the sample is washed with Triton X-100 reduced, treated with Dispase II, treated with benzonuclease, and washed with DPBS. Between each of these steps, the sample may be briefly rinsed for 5 minutes with a buffered saline solution such as PBS or DPBS using 3 times the volume of each solution in the step.

[0057] Optionally, the isolated ECM after nuclease treatment and washing can be stored, dialyzed, disinfected, sterilized, micronized, dehydrated (lyophilized), and / or converted into other forms for appropriate use. The isolated ECM can be stored at -20°C after nuclease treatment. The isolated ECM can also be stored in 10% formalin neutral buffer.

[0058] The isolated ECM can be sterilized by using gamma irradiation, electron beam (e-beam), glutaraldehyde, ethylene oxide, peracetic acid (PAA), ethanol, or CO2-based technologies. In embodiments, the isolated ECM is sterilized using CO2-based technologies that can sterilize soft materials without compromising the efficacy of the material. The isolated ECM can be micronized by sieving, freeze-grinding, mincing, spray drying, jet milling, or supercritical fluid (SCF) technologies. The isolated ECM can also be freeze-dried to remove water at low temperatures, which can be accomplished in a desiccator or low-temperature vacuum oven.

[0059] The isolated ECM can be micronized to a particle size of about 1-1000 microns, 1-900 microns, 1-800 microns, 1-700 microns, 1-600 microns, 1-500 microns, 1-400 microns, 1-300 microns, 1-200 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, or 100-125 microns. In embodiments, the particle size is about 100-500 microns. The micronized particles can be immediately dehydrated by lyophilization and stored at room temperature after lyophilization.

[0060] Lyophilization can include freezing under vacuum and drying at reduced pressure in one or more cycles at reduced pressure and low temperature. Exemplary temperatures for freezing isolated ECM for lyophilization can include -25°C to -60°C, -25°C to -50°C, -25°C to -45°C, -30°C to -45°C, -35°C to -45°C, or -45°C. Exemplary temperatures for drying isolated ECM samples for lyophilization can include 10°C to -50°C, 10°C to -45°C, 10°C to -40°C, 5°C to -40°C, 5°C to -30°C, 5°C to -25°C, 0°C to -25°C, -40°C, -25°C, or 5°C. Exemplary pressures for freezing and / or drying isolated ECM samples during lyophilization include 25mTorr to 375mTorr, 25mTorr to 360mTorr, 50mTorr to 360mTorr, 75mTorr to 360mTorr, 100mTorr to 360mTorr, 150mTorr to 325mTorr, 175mTorr to 300mTorr, 200mTorr to 300mTorr, 225mTorr to 275mTorr, 360mTorr, 300mTorr, 250mTorr, 100mTorr, or 50mTorr. The length of time for drying and / or freezing can range from 5 minutes to 800 minutes, 5 minutes to 750 minutes, 5 minutes to 720 minutes, 5 minutes to 400 minutes, 90 minutes to 350 minutes, 10 minutes to 720 minutes, 5 minutes, 120 minutes, 300 minutes, or 720 minutes. In embodiments, the isolated sample is freeze-dried in a first step by freezing at 360 mTorr for 5 minutes, followed by a first phase of drying at 100 mTorr, -40°C for 120 minutes, 50 mTorr, -25°C for 300 minutes, and 250 mTorr, -25°C for 720 minutes, followed by a second phase of drying at 300 mTorr, 5°C for 120 minutes.

[0061] In embodiments, the isolated ECM is not micronized. Additionally, the isolated ECM can be dehydrated under mild conditions without micronization. Mild conditions include temperatures below 37° C., for example, between 2° C. and −20° C. Examples of mild conditions include between 8° C. and −20° C. and a vacuum of 300 mTorr to 500 mTorr. Another example of dehydration under mild conditions includes a vacuum of 360 mTorr at 15° C.

[0062] In embodiments, amphibian samples may be decellularized as described above using NLS as a detergent and dehydrated under mild conditions without microparticulation. In embodiments, the method for decellularizing a sample includes the steps of briefly rinsing a 5×5 cm amphibian sample with a buffer solution such as PBS or DPBS for 5 minutes at room temperature; subsequently washing the sample with NLS for 6 hours at 37° C. with shaking at 100 RPM; subsequently rinsing the sample with a buffer solution such as PBS or DPBS for 5 minutes at room temperature; subsequently treating the rinsed sample with a protease such as dispase for 90 minutes at 37° C. followed by briefly rinsing the protease-treated sample with a buffer solution such as PBS at room temperature; followed by treating the rinsed sample with a nuclease such as benzonase for 6 hours; followed by rinsing the nuclease-treated sample with a buffer solution such as PBS or DPBS at room temperature for 5 minutes; and then washing the nuclease-treated sample with a buffer solution such as PBS or DPBS at 37°C for 24 hours with shaking at 100 RPM.

[0063] After decellularization using NLS as described herein, the sample is dehydrated under mild conditions without micronization. Dehydration under mild conditions includes freeze-drying and air drying at low temperature in a convection oven. An example of dehydration under mild conditions includes freeze-drying at 15° C. and 360 mTorr for 12 hours. Mild conditions also include drying in a desiccator with a desiccant. In an embodiment, after dehydration under mild conditions, the isolated ECM can be stored at room temperature covered with parafilm.

[0064] In embodiments, the isolated ECM can be prepared into a variety of forms, for example, a powder containing micronized particles that can be reconstituted with water, a buffer solution, or any suitable liquid for use as a solution, paste, liquid, extract, cream, lotion, serum, emollient, ointment, gel, gelatin, hydrogel, dispersion, or emulsion.

[0065] The isolated ECM can also be gelatinized to form gelatin. Methods for gelatinizing the isolated ECM include dissolving the isolated ECM in a solution containing a weak acid and an endopeptidase at 37° C. or room temperature under continuous stirring (90-120 RPM) for 12-48 hours to form gelatin. Examples of weak acids that can be used include acetic acid, formic acid, benzoic acid, hydrofluoric acid, and phosphoric acid. The concentration of the weak acid can be from 0.001M to 0.1M, 0.005M to 0.1M, 0.01M to 0.1M, or 0.005M to 0.05M. Examples of endopeptidases that can be used include pepsin, trypsin, chymotrypsin, and elastase. The mass / volume (w / v) percentage (%) concentration of the endopeptidase can be from 0.01% to 1%, 0.05% to 1%, 0.1% to 1%, or 0.05% to 0.5%. After dissolution, the gelatin can be separated and collected by centrifugation or filtration.

[0066] In an embodiment, a method for gelatinizing isolated ECM includes dissolving the isolated ECM in 0.01 M acetic acid and 0.1% pepsin at 37° C. under continuous agitation at 100 RPM for 24 hours, centrifuging the dissolved isolated ECM at 10,000×g for 10 minutes, and obtaining a supernatant containing the gelatinized ECM in the form of gelatin.

[0067] After decellularization, the isolated ECM can be analyzed by staining for visualization of the structural integrity of the decellularized ECM and removal of cells by light microscopy, as well as quantification of the amount of DNA. Staining methods include hematoxylin and eosin staining (H&E staining) and DAPI (4'6-diamidino-2-phenylindole). Hematoxylin is a basic dye that stains acidic components of cells such as the nucleus, while eosin is an acidic dye that stains basic components of cells such as the cytoplasm. DAPI is a blue fluorescent DNA stain. Surprisingly, it was found that more than 70% of the cells were removed from the isolated ECM, yet the ECM construct remained intact (Figure 1A-F). Additionally, the novel method of decellularization described herein removed a significant amount of DNA from cells; isolated ECM contained less than 10 ng of DNA, which is approximately 10% of the total DNA in native ECM (see Figures 2A-B). Furthermore, the length of the residual DNA fragments in isolated ECM was less than 300 bp in length (Figures 3A-B), whereas the length of DNA in native ECM was greater than 250 kilobases (kbp) in length.

[0068] The present disclosure describes isolated ECMs that have greater than 70% of the cells (natural cells) removed, but that have the structural and / or functional architecture of natural amphibian ECMs. The isolated ECMs have greater than 99%, 95%-99%, 90%-99%, 71%-99%, 75%-95%, 80%-90%, or 80%-85% of the natural cells in the ECM removed. The isolated ECMs have greater than 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the natural cells in the ECM removed. The isolated ECMs contain less than 30% of the natural cells in the ECM. Isolated ECM has 1%-29%, 5%-25%, 10%-20%, or 15%-20% of the native cells in the ECM removed. Isolated ECM contains less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or 25% of the native cells in the ECM. Isolated ECM contains an undetectable amount of native cells in the ECM.

[0069] The amount of residual DNA in the isolated ECM described herein is less than 1 ng / ul (ng DNA per microliter of extract) or is only 1-10 ng / μl, 2-9 ng / μl, 3-8 ng / μl, 3-7 ng / μl, 3-6 ng / μl, 3-5 ng / μl, 3-4 ng / μl, 2-3 ng / μl, 4-5 ng / μl, 5-6 ng / μl, 6-7 ng / μl, 7-8 ng / μl, 2.5 ng / μl, 3 ng / μl, 3.5 ng / μl, 4 ng / μl, 4.5 ng / μl, 5 ng / μl, 5.5 ng / μl, 6 ng / μl, 6.5 ng / μl, 7 ng / μl, 7.5 ng / μl, or 8.0 ng / μl (ng DNA per microliter of extract). In contrast, native (naive) ECM contains DNA in an amount of 100ng / μl (DNA per microliter of DNA extract solution). Compared to native ECM, the amount of residual DNA in isolated ECM is less than 1% of the amount of DNA in native ECM, or only 1%-10%, 2%-9%, 2%-5%, 3%-8%, 3%-7%, 3%-6%, 3%-5%, 3%-4%, 2%-3%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, or 8.0% of the amount of DNA in native ECM. The amount of residual DNA in isolated ECM is undetectable.

[0070] The amount of DNA in the isolated ECM described herein is less than 20 ng / mg dry ECM, in embodiments, the amount of DNA in the isolated ECM is 10-19 ng / mg dry ECM, 10-18 ng / mg dry ECM, 10-17 ng / mg dry ECM, 10-16 ng / mg dry ECM, 10-15 ng / mg dry ECM, 19 ng / mg dry ECM, 18 ng / mg dry ECM, or 17 ng / mg dry ECM.

[0071] Furthermore, the residual DNA fragments in the isolated ECM are less than 300 bp in length (see FIG. 3). The residual DNA fragments in the isolated ECM are undetectable, less than 50 bp in length, or 50-300 bp in length, 60-290 bp in length, 70-280 bp in length, 80-270 bp in length, 90-260 bp in length, 50-250 bp in length, 100-250 bp in length, 100-240 bp in length, 100-230 bp in length, 100-220 bp in length, 100-210 bp in length, or 100-200 bp in length. In an embodiment, the residual DNA fragments in the isolated ECM are less than 250 bp in length. Thus, the length of the residual DNA fragments in the isolated ECM is substantially shorter than that of the natural ECM, which is longer than 250 kbp in length.

[0072] The decellularization process described herein provides isolated ECM with substantially intact stromal structures and biological activity. The isolated ECM has sufficient structural and functional properties and / or reduced immunogenicity, and therefore it can be used as a biomaterial in vitro, ex vivo, and / or in vivo. The isolated ECM has a substantially native structure on which cells can grow. The isolated ECM can be used as a scaffold for cell attachment, proliferation, differentiation, regeneration, and tissue development in vitro, ex vivo, and / or in vivo without inducing any adverse effects. The isolated ECM is substantially free of immunogenic components, and therefore it can be administered to a subject. The isolated ECM is substantially free of cellular and nuclear debris, is substantially clean, and has reduced immunogenicity, and therefore it can be used as a biomaterial. The ECM is substantially free of cellular and nuclear debris, such as DNA.

[0073] The structural and functional properties of the native ECM are intact in the isolated ECM: components of the ECM for wound healing and treating and preventing skin conditions are retained in the isolated ECM.

[0074] The isolated ECM described herein is biologically active as shown by the results of bioassays, such as cell attachment assays, cell proliferation assays, and cell migration assays. Cell attachment assays measure adhesion between cells or between cells and surfaces or extracellular matrices, such as attachment and interaction with neighboring cells. Adhesion is important for various cellular processes, including growth and differentiation, which are also involved in wound healing. The results of the cell attachment assays are shown in Figures 5A, 5B, and 12, which confirm that the isolated ECM promoted cell attachment to various cells, such as skin cells, such as fibroblasts and HeCaT cells. In embodiments, the isolated ECM can be used to induce cell attachment and / or cell adhesion of cells.

[0075] Furthermore, Figures 5A and 5B show that decellularization with the NLS process (NLS wash with micronization and dehydration) is favorable for maintaining ECM protein bioactivity, and that the T2 process (Triton wash with dehydration without micronization) appears to improve the bioactivity of extracted ECM proteins. Figure 12 shows that the combination of NLS with mild dehydration without micronization improved cell attachment compared to the control. Thus, Figure 12 confirms that the combination of NLS with mild dehydration (without micronization) further improves the retention of protein bioactivity, as shown by the results of the attachment assay.

[0076] Cells grow by replication and division, which changes the number of cells in a population. Cell proliferation assays are used to determine cell growth by detecting changes in cell number. The results of cell proliferation assays are shown in Figures 6A, 6B, and 13, which confirm that isolated ECM promoted the proliferation of various cells, e.g., skin cells such as fibroblasts and HeCaT cells. In embodiments, isolated ECM can be used to induce cell proliferation.

[0077] Furthermore, Figures 6A and 6B show that the NLS process appears to promote and sustain proliferation to a greater extent for HeCaT cells than any other process. Figure 13 confirms that the combination of the NLS process with gentle dehydration (without micronization) improved proliferation for both fibroblasts and HeCaT cells.

[0078] Cell migration plays an important role in the development and maintenance of multicellular organisms. It is involved in cellular processes including tissue formation during embryonic development, wound healing, and immune response, all of which require the movement of cells in a specific direction to a specific location. Cells migrate in response to a signal, such as a chemical or mechanical signal. The results of the cell migration assay are shown in Figures 7A, 7B, and 14, which confirm that the isolated ECM induced cell migration of various cells, for example, skin cells such as fibroblasts and HeCaT cells. In an embodiment, the isolated ECM can be used to induce cell migration.

[0079] Furthermore, Figures 7A and 7B show that the T2 process appears to produce the greatest effect on cell migration for both fibroblasts and HeCaT cells. Figure 14 confirms that the process of combining NLS with mild dehydration without micronization showed a significant effect on both fibroblasts and HeCaT cells. The most significant improvement was shown for fibroblasts when compared to other decellularization methods.

[0080] In embodiments, the isolated ECM can induce various cells, such as skin cells, to have adhesion, proliferation, and migration activity. Examples of skin cells include fibroblasts, keratinocytes, melanocytes, Langerhans cells, endothelial cells, chondrocytes, muscle cells, bone cells, hair follicle epithelial stem cells, and Merkel cells.

[0081] The present disclosure describes ECM compositions and composites that include isolated ECM. In addition to isolated ECM, the composition may include one or more carriers or excipients. Examples of carriers and excipients include saline, emulsions, mixtures of organic solvents with water, calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, petrolatum, lanolin, mineral oil, dimethicone, humectants, and polyethylene glycol. Examples of humectants include glycerin, lecithin, and propylene glycol. In embodiments, the compositions described herein include cosmetic or pharmaceutical compositions that contain one or more cosmetically or pharma- ceutically acceptable carriers or excipients, respectively.

[0082] The compositions described herein may include one or more agents, such as one or more therapeutic agents, including known drugs such as retinoic acid, corticosteroids, antifungal agents, antiviral agents, antibiotics, antiseptics, local anesthetics, and anti-neoplastic agents.

[0083] The compositions described herein may include one or more agents, such as one or more cosmetic agents. Examples of cosmetic agents include antioxidants, peptides, alpha or beta hydroxy acids, retinol, vitamins, plant extracts, skin clarifying agents such as arbutin, moisturizers such as hyaluronic acid, emollients, carbohydrates, glycoproteins, and / or polymers. The one or more agents may include a combination of agents. The agents may be exogenous or heterologous to the isolated ECM.

[0084] Examples of one or more peptides and proteins include growth factors, cytokines, and chemokines. Examples of growth factors include fibroblast growth factors (FGFs), including acidic FGF, basic FGF, FGF8, and FGF10; ciliary neurotrophic factor (CNTF); epidermal growth factor (EGF); granulocyte macrophage colony-stimulating factor (GM-CSF); hepatocyte growth factor (HGF); insulin-like growth factors 1 and 2 (IGF-1 and IGF-2); keratinocyte growth factor (KGF); nerve growth factor (NGF); neurotrophins, such as neurotrophin-3, neurotrophin-4, and neurotrophin-5; platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); stromal cell-derived factor 1 alpha (SDF-1 alpha); and transforming growth factor-alpha and -beta (TGF-α and TGF-β). Examples of cytokines and chemokines include tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha and beta (IL-1α and IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-18 (IL-18), CCL2, CCL3, CCL5, CXCL1, CXCL4, CXCL5, CXCL7, CXCL8, and CXCL12. Examples of cosmetic peptides include acetyl hexapeptide, acetyl tetrapeptide, palmitoyl pentapeptide, and palmitoyl oligopeptide.

[0085] Examples of the one or more therapeutic agents include antibacterial agents and anti-inflammatory agents. Examples of antibacterial agents include antibiotics such as penicillin, streptomycin, amoxicillin, cephalexin, clindamycin, dicloxacillin, and doxycycline. Other antibacterial agents include antibacterial peptides, silver salts, clotrimazole, miconazole, and ketoconazole. Examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylic acid, ibuprofen, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, and sodium salicylamide.

[0086] Examples of cosmetic agents include one or more glycoproteins, including proteoglycans, which are proteins covalently attached to glycosaminoglycans (GAGs), antioxidants, ascorbic acid, vitamin C, alpha hydroxy acids (AHAs), beta hydroxy acids (BHAs), exfoliants, skin whitening agents, light diffusers, UV absorbers, sunscreens, moisturizers, anti-wrinkle ingredients, and oil absorbents. Examples of AHAs include glycolic acid, lactic acid, malic acid, tartaric acid, and citric acid. Examples of BHAs include salicylic acid.

[0087] The compositions described herein may also include one or more natural and / or synthetic polymers. Natural polymers may be derived from animal or non-animal sources, such as plant sources. Examples of natural polymers include natural polymers such as collagen, chitosan, alginate, glycosaminoglycans, fibrin, and hyaluronic acid. Examples of synthetic polymers include polyethylene, polyethylene glycol (PEG), polyethylene terephthalate (PET or PETE), polytetrafluoroethylene (PTFE), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), poly(ethylene glycol) diacrylate (PEG diacrylate), poly(hydroxy acid), polydioxanones, polycaprolactones, poly(orthoesters), polyanhydrides, polyphosphazenes, poly(amino acids), pseudopoly(amino acids), conductive polymers (e.g., polyacetylene, polypyrrole, polyaniline), polyurethanes, polystyrene, and nitinol.

[0088] The polymers of the compositions described herein may be biocompatible, biodegradable, and / or bioabsorbable and may be random copolymers, block copolymers, or blends of monomers, homopolymers, copolymers, and / or heteropolymers containing these monomers. Exemplary biodegradable or bioabsorbable polymers include polylactide, polyglycolide, polycaprolactone, polydioxane, and random and block copolymers thereof. The biodegradable and / or bioabsorbable polymers may contain monomers selected from the group consisting of glycolide, lactide, dioxane, caprolactone, trimethylene carbonate, ethylene glycol, and lysine. The biodegradable and / or bioabsorbable polymers may include bioabsorbable and biodegradable linear aliphatic polyesters such as polyglycolide (PGA) and its random copolymers, poly(glycolide-co-lactide-) (PGA-co-PLA). Other examples of suitable biocompatible polymers include polyhydroxyalkylmethacrylates, ethyl methacrylate, polyvinylpyrrolidone, and polyacrylamides. Other suitable bioabsorbable materials are biopolymers including collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acids, polylysine, and copolymers of these materials. Any combination of the above example polymers and copolymers or blends may also be included in the composition.

[0089] The compositions described herein may also include protectants, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffers, solubilizers, skin penetration enhancers, and surfactants.

[0090] Any skin penetration enhancer may be added to the composition, provided that the skin penetration enhancer is safe and capable of effectively promoting passage of the desired substance in the isolated ECM across the skin membrane. Examples of skin penetration enhancers include dimethylsulfoxide (DMSO), monoglycerides, C10-C20 fatty acid esters including ethyl palmitate and isopropyl myristate; caproyl lactylic acid and lauroyl lactylic acid. dodecyl (lauryl) acetate; lactate esters such as lauryl lactate and myristyl lactate; monoalkyl ethers of polyethylene glycols and their alkyl or aryl carboxylate esters, and carboxymethyl ethers such as polyethylene glycol-4 lauryl ether (Laureth-4) and polyethylene glycol-2 lauryl ether (Laureth-2); Myreth-3, myristyl sarcosine, and methyl laurate; polypropylene glycol, polyethylene glycol, lecithin, urea, amino acids, 1-dodecylhexahydro-2H-azepin-2-one (Azone), oleic acid, linoleic acid, isopropyl linoleate, oleyl alcohol, 1-dodecyl-azacycloheptan-2-one, butanediol, and 2-(2-ethoxyethoxy)ethanol (Transcutol).

[0091] The isolated ECM and compositions described herein can be prepared as dry powders, solutions, pastes, liquids, extracts, creams, lotions, serums, emollients, ointments, dispersions, gels, hydrogels, gelatinized compositions, or emulsions. The isolated ECM and compositions can be prepared into a variety of suitable shapes and sizes as they can be formed, layered, homogenized, and reconstituted. They can be formed into two-dimensional or three-dimensional shapes. They can be formed into sheets, meshes, grafts, plugs, or any shape or form for use. The sheets can include a backing, with or without an adhesive. The backing can be biodegradable or non-biodegradable. Two or more sheets can be layered together or slightly attached. The sheets can be oriented in the same direction, different directions, or at an angle. There can be 2 to 15 layers of sheets. The sheets can be from different sources of amphibian ECM.

[0092] In embodiments, the powder containing the micronized particles can be reconstituted with water, a buffer solution, or any suitable liquid and / or carrier to form a solution, paste, liquid, extract, cream, lotion, serum, emollient, ointment, dispersion, or emulsion. The solution can be a buffer solution similar to that used for the decellularization process.

[0093] The isolated ECM described herein has been shown to have therapeutic or cosmetic activity depending on the degree of decellularization, concentration, and / or final composition. Thus, the isolated ECM and therapeutic compositions described herein can be used in vitro, ex vivo, and / or in vivo. Because the isolated ECM has various biological activities involved in regenerative cell processes, the isolated ECM and compositions described herein can be used as biomaterials for wound healing, tissue regeneration, scaffolds for growing cells, and for treating and preventing various skin conditions. The biomaterials can be formed as implants for regenerating tissue. The isolated ECM can also act as a scaffold, so cells can grow on the ECM to enhance tissue and organ regeneration and wound healing. The biomaterials can also be applied to, coated on, and / or injected into implants or medical devices for introduction into a subject. The medical device can be a patch, bandage, or any suitable device for delivering the biomaterial. Thus, the biomaterials can be used as materials for any xenograft or xenograft.

[0094] The biomaterials can also serve as systems or devices for delivery of agents to tissues and organs to treat and / or prevent wound healing, tissue regeneration, and various skin conditions. Endogenous agents, such as growth factors, within the biomaterial can be delivered by immediate or controlled release. Additionally, exogenous or heterologous agents as described that are added to the biomaterial can be delivered by immediate or controlled release.

[0095] Carriers for immediate or sustained release preparations include polymers. The polymers can be biodegradable and / or bioabsorbable. By way of example, for controlled release, the biomaterials can be coated with polymers such as acrylic polymers, acrylic / methacrylic copolymers, cellulose acetate phthalate (CAP), Opadry®, and Ethocel®. For immediate release, the biomaterials can be coated with cellulose polymers such as hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), methylcellulose (MC), and sodium carboxymethylcellulose (NaCMC); vinyl derivatives such as polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol (PVA), and polyvinyl alcohol-polyethylene glycol copolymer; acrylic polymers such as Eudragit®; or glycols such as polyethylene glycol.

[0096] The isolated ECM and compositions described herein can be prepared as formulations for treating and / or preventing various skin conditions, including inflammatory and cancerous skin conditions. The formulations can be in the form of a dry powder, solution, paste, liquid, extract, cream, lotion, serum, emollient, ointment, dispersion, gel, hydrogel, gelatin, or emulsion. The amount of isolated ECM in the formulation is 0.001%-5.0% w / v, 0.01%-4% w / v, or 0.05%-1.0% w / v. The formulations can be placed on or impregnated into a dressing, such as a non-stick bandage, for application to a subject.

[0097] Examples of skin conditions requiring therapeutic treatment include acne, actinic keratosis, blisters, cellulitis, cold blisters, hives, impetigo, keratosis pilaris, liver spots, moles, ringworm, uticaria, vitiligo, and warts. Examples of inflammatory skin conditions include psoriasis; dermatitis such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, and lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea, and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. Examples of cancerous skin conditions include basal cell carcinoma, melanoma, and squamous cell carcinoma. Other skin conditions requiring treatment include fine lines and / or wrinkles, aging, redness, abrasions, burns, cuts, infections, razor burns, scars, uneven skin tone, sores, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. Burns include acute burns, including first, second, or third degree burns.

[0098] The isolated ECM and compositions described herein can also be used in skin care regimens to protect the skin from damage, including UV radiation and environmental pollution, and as aesthetic agents to enhance the appearance of the skin.

[0099] The isolated ECM and compositions described herein can be administered locally to a target site, or by injection, implantation, microneedling, or radiofrequency microneedling, or using an ablative fractional laser. The isolated ECM and compositions described herein can be administered to a subject before, during, or after a dermatological or cosmetic procedure, such as skin ablation, microdermabrasion, and ablative laser resurfacing. A dermatological or cosmetic procedure includes a procedure in which at least one cell of the stratum corneum is removed. The isolated ECM and compositions described herein can also be delivered as an injectable or with a dermal or subdermal implant, such as a volume filler, hyaluronic acid, or other dermal matrix protein, such as collagen or elastin, either naturally occurring, bioengineered, or recombinantly produced. The isolated ECM and compositions described herein can be administered alone or in combination with one or more agents described herein, such as growth factors, peptides, and proteins. The isolated ECM can also be administered with a toxin, such as botulinum toxin.

[0100] The isolated ECM and compositions described herein can reduce inflammation, reduce scarring, reduce keloid formation, reduce or lessen the severity of scarring and keloid formation, and / or reduce healing time for a variety of dermatological and cosmetic procedures. The isolated ECM and compositions described herein can also be used to restore lost dermal matrix or subdermal volume.

[0101] The present disclosure describes kits that include the isolated ECM or compositions described herein for various uses described herein. The kits include sterile isolated ECM or compositions thereof in any shape and form. The kits may include solutions for reconstituting the ECM for use. The kits may include devices for administering the isolated ECM or compositions to a subject. The kits may include implants that may be coated with the ECM or compositions prior to implantation in a subject. The kits may include components for various uses described herein.

[0102] The present disclosure also describes medical devices that include the isolated ECM or compositions described herein for various uses as described herein. A medical device can be a material or object that is used directly or indirectly to apply the isolated ECM or compositions described herein. As an example, a medical device can be used to apply the isolated ECM or compositions described herein onto the skin of a subject.

[0103] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of example language (e.g., "etc."), are intended merely to more fully clarify the invention and do not pose limitations on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0104] Numbers expressing ranges or amounts of ingredients, constituents, reaction conditions, and the like used in the specification and claims should be understood to be modified by the term "about." Where further clarity is needed, the term "about," when used in conjunction with a stated numerical value or range, shall have the meaning that would be reasonably ascribed to it by one of ordinary skill in the art, i.e., slightly more or slightly less than the stated value or range, within ±20% of the stated value; ±15% of the stated value; ±10% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; ±1% of the stated value; or within any percentage range between 1% and 20% of the stated value.

[0105] As will be understood by those skilled in the art, each embodiment disclosed herein may comprise, consist essentially of, or consist of its specific stated elements, steps, ingredients, or components. Thus, the terms "include" or "including" should be interpreted as reciting "comprises, consists of, or consists essentially of." The transitional words "comprise" or "comprises" mean "including but not limited to," allowing for the inclusion of unspecified elements, steps, ingredients, or components, even in large amounts. The transitional phrase "consisting of" excludes any unspecified elements, steps, ingredients, or components. The transitional phrase "consisting essentially of" limits the scope of an embodiment to the specified elements, steps, ingredients, or components and those that do not substantially affect the embodiment. In an embodiment, the lack of a substantial effect of a step is evidenced by the lack of a statistically significant decrease in the process step of removing cellular debris, such as DNA, from the sample. Lack of substantial effect of an embodiment may include a lack of statistically significant improvement in using the isolated ECM in wound healing, cell growth, cell repopulation, cell attachment, cell proliferation, or cell migration.

[0106] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand way of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. The description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. as well as individual numbers within that range, for example, 1, 2, 2.5, 2.7, 3, 4, 5, 5.1, 5.3, 5.8, and 6. Moreover, any ranges mentioned herein are inclusive.

[0107] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations: each group member may be referred to or claimed individually or in any combination with other members of the group or other elements found herein.

[0108] The following exemplary embodiments and examples illustrate exemplary methods provided herein. These exemplary embodiments and examples are not intended, nor should they be construed, as limiting the scope of the present disclosure. It will be apparent that the method can be practiced otherwise than as specifically described herein. Numerous modifications and variations are possible in light of the teachings herein, and therefore are within the scope of the present disclosure.

[0109] Exemplary embodiments The following are exemplary embodiments: 1. A gelatinized ECM in the form of gelatin comprising isolated amphibian ECM, the isolated amphibian ECM being decellularized and containing intact native ECM structure, comprising: contain less than 1%–10% of the total DNA present in the corresponding native ECM sample; Contains residual DNA fragments smaller than 300 base pairs (bp); and / or More than 70% of the cells in the ECM are removed The gelatinized ECM is characterized by: 2. The gelatinized ECM of embodiment 1, wherein the amphibian ECM is characterized as having less than 1%, 2%-9%, 2%-5%, 3%-8%, 3%-7%, 3%-6%, 3%-5%, 3%-4%, 2%-3%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 1.0%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, or 8.0% of the total amount of DNA present in a corresponding native ECM sample. 3. The gelatinized ECM of embodiment 1 or 2, wherein the amphibian ECM is characterized as having residual DNA fragments less than 50 bp in length, 50-300 bp in length, 60-290 bp in length, 70-280 bp in length, 80-270 bp in length, 90-260 bp in length, 100-250 bp in length, 100-240 bp in length, 100-230 bp in length, 100-220 bp in length, 100-210 bp in length, or 100-200 bp in length. 4. A gelatinized ECM described in any one of embodiments 1 to 3, wherein the amphibian ECM is characterized by having residual DNA fragments less than 250 bp in length. 5. A gelatinized ECM described in any one of embodiments 1 to 4, wherein the amphibian ECM is characterized by having more than 99%, 95% to 99%, 90% to 99%, 71% to 99%, 75% to 95%, 80% to 90%, or 80% to 85% of the cells in the ECM removed. 6. The gelatinized ECM of any one of embodiments 1 to 5, wherein the ECM is obtained from a juvenile amphibian, and optionally the juvenile amphibian includes a froglet, a tadpole, an urodela, or a larval stage of an juvenile Apoda. 7. A gelatinized ECM described in any one of embodiments 1 to 6, wherein the ECM is obtained from a juvenile mature urodele. 8. A composition comprising a gelatinized ECM according to any one of embodiments 1 to 7, or a gel or hydrogel comprising an isolated amphibian ECM according to any one of embodiments 1 to 7, and a carrier. 9. The composition of embodiment 8, wherein the composition is a pharmaceutical or cosmetic composition, optionally, a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a cosmetic composition comprising a cosmetically acceptable carrier. 10. The composition of embodiment 8 or 9, wherein the composition further comprises one or more active substances, optionally wherein the one or more active substances are heterologous to the amphibian ECM. 11. The composition of any one of embodiments 8 to 10, further comprising one or more active agents which are peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or combinations thereof. 12. The composition of any one of embodiments 8 to 11, further comprising one or more growth factors, cytokines, chemokines, or combinations thereof. 13. The composition of any one of embodiments 8 to 12, wherein the composition further comprises one or more polymers, optionally wherein the one or more polymers are synthetic or natural polymers or copolymers. 14. A method for decellularizing amphibian ECM, comprising the steps of: obtaining a biological sample from an amphibian; washing the sample with a detergent solution containing an anionic surfactant; treating the sample with one or more proteases and one or more nucleases to obtain isolated ECM; and dehydrating the isolated ECM under mild conditions. 15. The method of embodiment 14, comprising the steps of: collecting a biological sample from an amphibian; rinsing the sample with a solution; washing the sample with a detergent solution comprising an anionic surfactant; treating the sample with a protease; rinsing the sample after protease treatment; and treating the rinsed protease-treated sample with a nuclease to obtain isolated ECM; and dehydrating the isolated ECM under mild conditions; and optionally further washing the isolated ECM prior to the dehydration step. 16. The method of embodiment 14 or 15, comprising rinsing with water or a buffer solution, optionally phosphate buffered saline (PBS) or Dulbecco's phosphate buffered saline (DPBS). 17. The method of any one of embodiments 14 to 16, wherein the sample is washed with a detergent solution comprising N-lauryl sarcosine (NLS), potassium lauryl sarcosine, cholate, deoxycholate, sodium dodecyl sulfate (SDS), or polyoxyethylene sorbitan fatty acid esters, optionally Tween 20, 40, 60, or 80. 18. A method according to any one of embodiments 14 to 17, wherein the sample is incubated with one or more proteases, such as one or more aminoendopeptidases. 19. The method of any one of embodiments 14 to 18, wherein the sample is incubated with one or more proteases, such as dispase I, dispase II, trypsin, papain, and collagenase. 20. A method according to any one of embodiments 14 to 19, wherein the sample is incubated with one or more nucleases, such as non-specific DNA / RNA endonucleases. 21. The method of any one of embodiments 14 to 20, wherein the sample is incubated with one or more nucleases, such as benzonuclease, turbonuclease, pulmozyme, and a combination of deoxyribonuclease and ribonuclease, optionally a combination of recombinant deoxyribonuclease and ribonuclease. 22. The following steps are carried out in the following order: obtaining a biological sample from the amphibian; rinsing the sample with buffered saline; washing the sample with a detergent solution containing NLS; rinsing the sample with buffered saline; treating the rinsed sample with Dispase II; rinsing the sample with buffered saline; treating the rinsed sample with benzonuclease to obtain isolated ECM; and A process to dehydrate samples under mild conditions 22. The method of any one of embodiments 14 to 21, wherein the isolated ECM is optionally washed with DPBS prior to the dehydration step under mild conditions. 23. The method of any one of embodiments 14 to 22, wherein the step of dehydrating under mild conditions comprises dehydrating under a reduced pressure of 360 Torr at 15°C. 24. A method according to any one of embodiments 14 to 23, wherein the sample is washed with detergent solution for 6 hours with shaking. 25. A method according to any one of embodiments 14 to 25, wherein the sample is rinsed with DPBS for 5 minutes between the steps of washing with detergent solution and treating with protease, and between the steps of treating with protease and treating with nuclease. 26. The method of any one of embodiments 14 to 25, wherein the amphibian comprises a froglet, a tadpole, an urodela, or a larval stage of an Apoda. 27. The method of any one of embodiments 14 to 26, wherein the biological sample comprises ECM tissue from a juvenile urodele. 28. The method of any one of embodiments 14 to 27, wherein the biological sample comprises ECM of connective tissue, adipose tissue, bone, plasma, skin, cartilage, tendon, dura, or fascia. 29. An isolated ECM obtained by a method according to any one of embodiments 14 to 28. 30. A method of gelatinizing isolated ECM, comprising dissolving the isolated ECM in a solution containing a weak acid and an endopeptidase to form gelatin. 31. The isolated ECM comprises a decellularized amphibian ECM that contains an intact native ECM structure, comprising: contain less than 1%–10% of the total DNA present in the corresponding native ECM sample; Contains residual DNA fragments smaller than 300 base pairs (bp); and / or More than 70% of the cells in the ECM are removed 31. The method of embodiment 30, characterized by: 32. The method of embodiment 30 or 31, wherein the isolated ECM is characterized as having less than 1%, 2%-9%, 2%-5%, 3%-8%, 3%-7%, 3%-6%, 3%-5%, 3%-4%, 2%-3%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 1%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, or 8.0% of the total amount of DNA present in the corresponding native ECM sample. 33. The method of any one of embodiments 30 to 32, wherein the isolated ECM is characterized as having residual DNA fragments of 50 to 300 bp in length, 60 to 290 bp in length, 70 to 280 bp in length, 80 to 270 bp in length, 90 to 260 bp in length, 100 to 250 bp in length, 100 to 240 bp in length, 100 to 230 bp in length, 100 to 220 bp in length, 100 to 210 bp in length, or 100 to 200 bp in length. 34. A method according to any one of embodiments 30 to 33, wherein the isolated ECM is characterized as having residual DNA fragments less than 250 bp in length. 35. The method of any one of embodiments 30 to 34, wherein the isolated ECM is characterized by having 71% to 99%, 75% to 95%, 80% to 90%, or 80% to 85% of the cells in the ECM removed. 36. The method of any one of embodiments 30 to 35, wherein the isolated ECM is obtained from a juvenile amphibian, and optionally the juvenile amphibian comprises a froglet, a tadpole, an urodela, or a larval stage of an juvenile Apoda. 37. A method according to any one of embodiments 30 to 36, wherein the isolated ECM is obtained from a juvenile urodele. 38. The method of embodiment 30 or 31, wherein the isolated ECM comprises the isolated ECM of embodiment 29. 39. Gelatinized ECM in the form of gelatin, obtained by a method according to any one of embodiments 31 to 38. 40. A composition comprising the gelatin of embodiment 39, or a gel or hydrogel comprising the isolated ECM of embodiment 29. 41. The composition described in embodiment 40, wherein the composition is a pharmaceutical or cosmetic composition, optionally, a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a cosmetic composition comprising a cosmetically acceptable carrier. 42. The composition of embodiment 40 or 41, wherein the composition further comprises one or more active substances, optionally wherein the one or more active substances are heterologous to the isolated ECM. 43. The composition of any one of embodiments 40 to 42, further comprising one or more active agents which are peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or combinations thereof. 44. The composition of any one of embodiments 40 to 43, further comprising one or more growth factors, cytokines, chemokines, or combinations thereof. 45. The composition of any one of embodiments 40 to 44, wherein the composition further comprises one or more polymers, optionally wherein the one or more polymers are synthetic or natural polymers or copolymers. 46. ​​A method for treating and / or preventing a skin condition, comprising administering to a subject in need thereof a gelatinized ECM described in any one of embodiments 1-7 or 39, or a composition described in any one of embodiments 8-13 or 40-45. 47. The method of embodiment 46, wherein the skin conditions include fine lines and / or wrinkles, aging, redness, abrasions, burns, cuts, infections, razor burns, scars, uneven skin tone, soreness, stretch marks, increasing skin elasticity and / or firmness, improving skin hydration, inflammation, and hyperpigmentation. 48. The method of embodiment 46 or 47, wherein preventing the skin condition comprises protecting the skin from damage including UV radiation and / or environmental pollution. 49. The method of any one of embodiments 46 to 48, wherein treating and / or preventing skin conditions includes skin conditions following dermatological or cosmetic procedures, optionally wherein the procedures include skin ablation, microdermabrasion, or ablative laser resurfacing. 50. The method of any one of embodiments 46-49, wherein treating and / or preventing a skin condition comprises improving the appearance of the subject's skin using a gelatinized ECM described in any one of embodiments 1-7 or 39, or a composition described in any one of embodiments 8-13 or 40-45. 51. A formulation comprising a gelatinized ECM described in any one of embodiments 1 to 7 or 39, an isolated ECM described in embodiment 29, or a composition described in any one of embodiments 8 to 13 or 40 to 45. 52. The formulation of embodiment 51 in the form of a powder, solution, paste, liquid, extract, cream, lotion, serum, dispersion, emulsion, ointment, gel, hydrogel, or gelatin. 53. The formulation of embodiment 51 or 52, comprising 0.001% to 5.0% w / v ECM. EXAMPLES

[0110] Example 1 Collecting skin samples from axolotls Caudata skin samples are obtained or harvested from juvenile urodela. A 5 x 5 centimeter (cm) full thickness skin explant sample of skin is surgically removed from a juvenile urodela. The sample is cleaned and prepared for decellularization.

[0111] Example 2 Decellularization of harvested axolotl skin samples Fresh axolotl samples were imaged to document their pre-processing appearance. The mass of all samples was measured before and after each step to record mass loss due to decellularization (Table 1).

[0112] Washing. Three 5 x 5 cm samples were briefly rinsed with Dulbecco's Phosphate Buffered Saline (DPBS) for 5 minutes (min) at room temperature, followed by washing with Triton X-100 reduced or NLS for 6 hours at 37°C, and then briefly rinsed with DPBS for 5 minutes at 37°C before incubation with one or more proteases. Washing with detergent solution was performed with shaking at 100 RPM.

[0113] Triton X-100 reduced wash (samples T1 and T2): After rinsing with PBS, each 5 x 5 cm sample was washed twice with 20 ml of 0.5% v / v Triton X-100 reduced containing 0.02% w / v sodium azide at 37°C for 6 hours each time with shaking at 100 RPM.

[0114] NLS Wash (Sample NLS): After rinsing with PBS, each 5 x 5 cm sample was washed twice with 25 ml of 1.0% NLS containing 0.02% sodium azide at 37°C for 6 hours each time.

[0115] Protease treatment. Samples were each incubated with 2 units / ml Dispase II (2 units of Dispase II per ml of high glucose DPBS) for 90 minutes at 37° C. Protease-treated samples were each rinsed briefly with DPBS for 5 minutes at room temperature.

[0116] Nuclease treatment: After rinsing with DPBS, the protease-treated samples were then treated with 40 units / ml benzonuclease (40 units of benzonuclease per ml of DPBS) for 6 hours at 37° C. Samples were then rinsed briefly with DPBS for 5 minutes at room temperature and washed with DPBS for 24 hours at 37° C.

[0117] Example 3 Analysis of isolated ECM Hematoxylin and Eosin (H&E) Staining. After decellularization was completed, two 8 mm punch biopsies (one from the top left corner and one from the center of each 5 x 5 specimen) were taken from each of the three specimens to compare the success of the decellularization process based on anatomical region.

[0118] H&E slides were processed and imaged by light microscopy (Figure 1A-F). A degree of decellularization greater than 80% was achieved as assessed by visual inspection.

[0119] DNA quantification. To quantify DNA in isolated ECM after the decellularization process, 4 mm punch biopsy samples were cut from each of the three samples from two different locations (bottom left corner and center of each 5x5 sample). Immediately after cutting, the samples were transferred to Eppendorf tubes and frozen at -20°C until ready for DNA extraction and quantification. DNA was extracted and purified using the DNEasy Blood & Tissue Kit. Samples were subjected to the protocol included in the kit and then quantified with the NanoDrop One Instrument.

[0120] The results shown in the graphs of Figures 2A and 2B indicate the proper removal of DNA by benzonase treatment. In Figure 2A, samples collected from the center of the tissue (C) for T1 (T1_C), T2 (T2_C), and NLS (NLS_C) yielded DNA concentrations (ng of DNA per ul of DNA extract) of 4.44 ng / μl ± 0.9, 3.96 ng / μl ± 0.83, and 3.27 ng / μl ± 0.52, respectively. Samples collected from the bottom left corner (BLC) of T1, NLS, and T2 yielded DNA concentrations of 7.44 ng / μl ± 3.88, 5.07 ng / μl ± 0.61, and 4.3 ng / μl ± 0.48 for T1_BLC, T2_BLC, and NLS_BLC, respectively. Figure 2B shows the DNA concentrations converted to percentages.

[0121] DNA Gel Electrophoresis. DNA quantification was completed by gel electrophoresis and shown in Figure 3A and Figure 3B. Samples were prepared using Purple 6X gel loading dye (New England BioLabs, B7024S) at a 5:1 ratio of DNA:stain. A DNA ladder (GeneRuler Express DNA Ladder reference number SM1553) was used for reference. A total volume of 12 μL (10 μL DNA sample + 2 μL Purple dye) was used per well for samples and DNA ladder. Samples were run at 100 volts for 45 minutes using an agarose gel (1%) containing GelRed Nucleic Acid Stain (MilliporeSigma SCT123). The gel was then removed and imaged on an Azure Biosystems c150 gel imager. As shown in Figures 3A and 3B, faint DNA bands were observable in the samples tested, suggesting that the benzonase treatment was effective.

[0122] Example 4 Microparticulation of isolated ECM Freeze-milling. T1 and NLS samples were micronized by freeze-milling to obtain particles. Samples were freeze-milled in a Spex 6870 freezer / mill under the following conditions: First, the samples were flash frozen in liquid nitrogen without direct exposure. Then, 3 ml of RNA / DNA-free water (Thermo-Scientific, Water, Nuclease-Free Reference R0582) was added 1 ml at a time to the freeze-milling chamber containing the sample. Immediately afterwards, the bullet was introduced into the freeze-milling device and a total of 6 cycles were performed with 5 minutes pre-cooling, 1 minute freeze-milling, 2 minutes cooling, and 6 impacts per second. A small sample of the slurry was set aside for visual examination under optical microscopy. Additionally, 15 randomly selected micronized tissue samples were measured and tabulated by ImageJ to assess particle length (fiber-like structures) and area (patches). The Triton X-100-treated samples yielded a fiber length of 28.36 μm ± 13.19 compared to 30.05 μm ± 34.70 for the NLS-treated samples (FIG. 4A). The average area of ​​the micronized T1 and NLS samples was 494.06 μm, respectively. 2 ±293.2 and 631.5μm 2 ±550.91. Furthermore, the fiber size distribution was more uniform in the Triton X-100 treated samples than in the NLS treated samples. The results provide evidence that the freeze-grinding process and settings are effective in microparticulating the axolotl decellularized tissue to lengths less than 100 μm.

[0123] The T2 sample was not micronized but was dehydrated under reduced pressure at 15° C. and 360 Torr.

[0124] Lyophilization. Immediately after freeze-grinding, the T1 and NLS samples were transferred to 50 ml conical tubes for freeze-drying. The process for freeze-drying was as follows: the samples were frozen at -45°C for 5 minutes at 360 Torr. Then, the samples underwent freezing at -40°C for 120 minutes at 100 mTorr, followed by freezing at -25°C for 300 minutes at 50 mTorr, and freezing at -25°C for 720 minutes at 250 mTorr. Finally, the samples underwent a second drying phase involving cooling at +5°C for 120 minutes at 300 mTorr.

[0125] The above subject matter is provided by way of example only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without departing from the true spirit and scope of the present disclosure, the true spirit and scope of which is set forth in the following claims, without following the example embodiments and applications illustrated and described.

[0126] Example 5 Gelatinization of samples T1, T2, and NLS Gelatinization. Gelatinization of decellularized and dehydrated isolated ECM samples (T1, T2, and NLS) was performed under the following conditions and tested for protein content by bicinchoninic acid (BCA) protein quantification assay. Briefly, samples were weighed to make a 1% w / v solution of isolated ECM in the following solvents: 0.01 M acetic acid and 0.1% pepsin. Samples were dissolved for 24 hours at 37° C. under stirring (100 RPM). After dissolution, samples were centrifuged at 10,000×g for 10 minutes and the supernatant was collected and used for bioassays. Any acidic solutions were neutralized with a base such as sodium hydroxide before testing or use in processing.

[0127] Bioassays. Bioassays were performed to quantify the activity of proteins in isolated ECM samples after gelatinization. All of the assays were performed on HeCaT cells and fibroblasts using crystal violet as a colorimetric measurement agent, except for the migration assay, which was evaluated by image processing.

[0128] Adhesion assay: All gelatinized samples, T1, T2, and NLS, were tested for cell attachment on both HeCaT cells and fibroblasts under the following conditions. Briefly, 24-well non-culture treated plates were coated with 32 μl, 64 μl, or 128 μl of gelatinized sample. Wells were left to be coated overnight at 2-8°C, subsequently washed once with PBS, and air-dried under a biosafety cabinet (BSC). Immediately after air-drying, 50,000 cells were added in a 50 μl volume per well and topped up with 950 μl of medium for a total volume of 1 ml. Cells were allowed to attach for 4 hours, after which they were fixed and stained simultaneously with a crystal violet / methanol solution (0.5% crystal violet in a 20:80 H2O:methanol solution). The crystal violet / methanol solution was allowed to infiltrate the cells for 20 minutes at room temperature under continuous agitation. Extraction of crystal violet was accomplished with lysis buffer for 30 minutes. Plates were read at 570 nm wavelength using a plate reader.

[0129] The attachment assay results suggest that all three samples (T1, T2, and NLS) treated with gelatinization maintained ECM protein bioactivity. A significant difference in attachment density is evident from the images compared to the bare well positive control group. The quantified results are shown in Figure 5A and Figure 5B, which provide further evidence that all samples performed better than the bare well control for both HeCaT and fibroblast cells. However, while all groups showed positive preliminary results, both T2 and NLS demonstrated better performance than T1 for both cell types. Furthermore, HeCaT cells appear to be more sensitive to axolotl ECM proteins than fibroblast cells overall. Data from the attachment assay suggest that all three methods adequately decellularized the ECM, but for determining the best method for decellularization, at any concentration, T2 may be best, followed by NLS, followed by T1. Additionally, the 64 μl coating appears to perform just fine, or even better than the 128 μl coating. It must be noted that a direct comparison of T2 with NLS or T1 is not appropriate, since T2 underwent a different dehydration protocol than T1 and NLS. Furthermore, T2 was not subjected to micronization by freeze-grinding. Nevertheless, the NLS process appears to be advantageous in maintaining ECM protein bioactivity, and the dehydration process of T2 appears to have improved the bioactivity of the extracted ECM proteins. For this reason, the combination of NLS with mild dehydration without micronization may further improve the retention of protein bioactivity as per the adhesion assay.

[0130] Proliferation assay: Proliferation assays were performed on both types of cells, HeCaT cells and fibroblasts, at a protein coating concentration of 64 μl (i.e., 1:1 dilution in PBS). Briefly, 96-well plates were left to be coated overnight at 2-8°C, then washed once with PBS and subsequently air-dried under a BSC. Wells were seeded with 10,000 cells in a final total volume of 200 μl. All samples were performed in triplicate and assessed by optical density (OD) measurements at 570 nm wavelength at 24, 48, and 72 hour intervals.

[0131] Results from the proliferation assay suggest enhanced cell proliferation for all samples and cell types. Nevertheless, HeCaT cells appear to produce a more pronounced effect than fibroblasts overall. Furthermore, in HeCaT cells, NLS-treated ECM appears to promote and sustain proliferation to a greater extent than any other group, producing higher OD readings (2.032±0.331) even at 72 hours, where all other groups (including control (0.912±0.011)) show attenuation (FIGS. 6A and 6B). Thus, the proliferation data confirm that the combination of NLS decellularization with T2 gentle dehydration without micronization can further preserve bioactivity and improve proliferation outcomes for fibroblasts and HeCaT cells.

[0132] Migration: Migration assays were performed in non-culture treated 6-well plates with ibidi® inserts. Wells were coated overnight at 2-8°C with the same concentrations and conditions as in the proliferation assay. Similarly, wells were washed once with PBS and air-dried under BSC. After complete drying, wells were fitted with ibidi® inserts as per the manufacturer's instructions. 20,000 cells were then seeded on each side of the ibidi® insert to ensure cell confluency within the chamber of the insert. Cells were left to attach and grow for 24 hours before removing the ibidi® insert. Immediately after removal of the ibidi® insert, images were taken as a baseline measurement and subsequently at three imaging time points (1:30 PM, 3:50 PM, and 8:00 AM). Images were processed using Fiji (ImageJ) and their intensity values ​​were measured across the empty space left by removing the ibidi® insert. This gap was measured periodically over the three time points and closure was calculated (see Figures 7A and 7B).

[0133] Evidence of enhanced migration was demonstrated for HeCaT cells, but fibroblast migration did not appear to be enhanced over time. At 8:00 a.m., fibroblasts in the control group (120.24 ± 17.852) produced completely closed gaps compared to the T1 (35.903 ± 4.737), T2 (46.370 ± 11.482), and NLS (42.197 ± 9.886) groups. On the other hand, at 8:00 a.m., HeCaT cells produced significant migration into the gap compared to the control group (6.731 ± 2.789) (T1: 11.625 ± 3.778, T2: 27.738 ± 3.054, and NLS: 16.695 ± 1.271). Overall, T2 appears to produce the most pronounced effects on cell migration for both HeCaT and fibroblast cells.

[0134] Results. Based on visual inspection, the overall degree of decellularization appears to fall within the defined acceptance criteria of greater than 80%. Nevertheless, differences in tissue integrity were observed and recorded for Triton 2 (T2) compared to the other samples. Triton 2 contained subdermal fat that was depicted on the H&E slides, which may explain its stable mass recorded throughout the experiment. Finally, the NLS-treated samples were more viscous and therefore more difficult to correctly measure mass.

[0135] With regard to benzonase treatment, the enzyme is able to remove over 90% of the genomic DNA of the ECM while maintaining the ECM integrity. There was a clear fading band in the lower region of the agarose gel, further supporting the conclusion that benzonase treatment effectively digests tissue genomic DNA. Furthermore, freeze-grinding and lyophilization resulted in particle sizes less than 100 μm long. Nevertheless, differences were observed between Triton- and NLS-treated tissues in the final product. Triton-treated tissues produced a more uniform particle size distribution (28.36 μm ± 13.19) when compared to NLS (30.05 μm ± 34.70). Observable differences in lyophilized yields were demonstrated. First, the T1 sample collapsed upon extraction from the lyophilizer due to inadequate drying during the second drying phase, and therefore an additional lyophilization at 250 mTorr for 30 min at 15°C was required to ensure complete dehydration of the tissue. On the other hand, NLS was completely lyophilized under the conditions specified above without disintegration.

[0136] Conclusion. The methods described herein for decellularizing, dehydrating, and gelatinizing axolotl ECM were successful in maintaining the bioactivity of extracted proteins. Based on the results from the bioassay, the new process combining NLS decellularization with the gentle dehydration method of T2 can be used to decellularize ECM samples. Furthermore, the data confirmed that HeCaT cells were more significantly affected by axolotl ECM proteins than fibroblasts. Nevertheless, fibroblasts were observed to perform better with T1 treatment than with T2 and NLS, which was the opposite of HeCaT cells, which performed better with T2 and NLS compared to fibroblasts.

[0137] Example 6 A new method for decellularization: Combining NLS with gentle dehydration Decellularization. A single 5x5cm axolotl skin sample was harvested as described in Example 1 and decellularized using the NLS process as described in Example 2 above. The mass of the sample was measured before and after every procedure during decellularization, and the results were tabulated and shown in Figure 8. After decellularization, the sample was dehydrated under gentle conditions as a whole sample without freeze-grinding / micronization. Immediately after decellularization, the sample was placed flat on a Petri dish and subjected to gentle dehydration at 15C and a vacuum of 360 Torr for 12 hours. Upon completion of the dehydration process, the sample was covered with parafilm and stored at room temperature until further processing.

[0138] H&E. After decellularization was completed, two 8 mm punch biopsies (one from the upper left corner and one from the center) were taken from the samples to compare the success of the decellularization process by anatomical region. Both samples were immediately stored in 10% formalin neutral buffer before shipping to the laboratory for H&E processing.

[0139] H&E slides were processed and imaged by light microscopy. A degree of decellularization above 80% was achieved as assessed by visual inspection (Figures 9A-C). The naive tissue sample appeared to have fewer cells and other anatomical markers than those found in the literature. The other two samples, from the center and top left corner, showed no visible cells or other anatomical features other than an intact extracellular matrix (ECM).

[0140] DNA quantification data. To quantify DNA after the decellularization process, 8 mm punch biopsy samples were extracted from two different locations (top left corner and center). After excision, samples were immediately transferred to Eppendorf tubes and frozen at -20°C until ready for DNA extraction and quantification. Additionally, after gentle dehydration, another 8 mm punch biopsy sample was taken from the center to quantify DNA per mg of dry mass of tissue. This sample recorded a mass of 19 mg. DNA was extracted and purified using the DNEasy Blood & Tissue Kit. Samples were subjected to the protocol included in the kit and then quantified with the NanoDrop One Instrument.

[0141] The amount of DNA in each sample is 24.29±0.007ng / ml (naive), 1.524±0.105ng / ml (NLS_TLC), and 1.772±0.186ng / ml (middle) (FIG. 10). Based on the dried specimen results, the amount of DNA in the samples is 17ng / mg of dried tissue. Additionally, the prior sample yielded over 100ng / ml, while the naive sample yielded 24.29±0.007ng / ml, which explains the lack of cells visibly present on the H&E processed sections.

[0142] Gel electrophoresis. DNA quantification was completed by gel electrophoresis to ensure that residual DNA fragments were less than 250 bp in length. Samples were prepared using Purple 6x gel loading dye (New England BioLabs, B7024S) at a 5:1 ratio of DNA:stain. A DNA ladder (GeneRuler Express DNA Ladder reference number SM1553) was also used for reference. A total volume of 12 μL (10 μL DNA sample + 2 μL Purple dye) was used per well for samples and DNA ladder. Samples were run at 100 volts for 45 minutes using an agarose gel (1%) containing GelRed Nucleic Acid Stain (MilliporeSigma SCT123). Gels were then removed and imaged on an Azure Biosystems c150 gel imager.

[0143] Figure 11 shows that there are no visible bands for the top left corner (TLC) and center of the dried sample (Dry). In comparison to the previous results, bands above 5 kbp can be observed for the naive tissue sample, suggesting that benzonase treatment removed DNA to acceptable levels.

[0144] Gelatinization. Gelatinization was performed as described in Example 5 above. Gelatinization was performed with 0.1% w / v pepsin in 0.01 M acetic acid at 37° C. under continuous stirring for 24 hours. After lysis, samples were centrifuged at 10,000×g for 10 minutes and the supernatant containing gelatinized isolated ECM was used for bioassays.

[0145] Bioassays. The following bioassays were performed to quantify the activity of ECM proteins after gelatinization of NLS-washed samples with gentle dehydration. All of the assays were performed on HeCaT cells and fibroblasts using crystal violet as a colorimetric agent, except for the migration assay, which was evaluated by image processing.

[0146] Adhesion assay: NLS samples with gentle dehydration were tested for cell attachment on both HeCaT cells and fibroblasts under the following conditions. Briefly, 24-well non-culture treated plates were coated with 32 μl, 64 μl, or 128 μl of gelatinized material. Wells were left to be coated overnight at 2-8°C, subsequently washed once with PBS, and air-dried under a BSC. Immediately after air-drying, 50,000 cells were added in a 50 μl volume per well and topped off with 950 μl of medium for a total volume of 1 ml. Cells were allowed to attach for 4 hours, after which they were fixed and stained simultaneously with a crystal violet / methanol solution (0.5% crystal violet in a 20:80 H2O:methanol solution). The crystal violet solution was allowed to infiltrate the cells for 20 minutes at room temperature under continuous agitation. Extraction of crystal violet was achieved with lysis buffer for 30 minutes and read at 570 nm wavelength using a plate reader.

[0147] This process did not result in significant differences in cell attachment correlated to protein plating concentration for both fibroblasts and HeCaT cells. Nevertheless, improved attachment can be observed when compared to the control group for both cell types. As shown in FIG. 12, the optical density values ​​for fibroblasts are 0.896±0.019, 0.872±0.059, 0.869±0.205, and 0.699±0.082 for 32μl, 64μl, 128μl, and control, respectively. On the other hand, the optical density values ​​for HeCaT cells are 0.432±0.029, 0.449±0.022, 0.427±0.027, and 0.227±0.04 for 32μl, 64μl, 128μl, and control, respectively (see FIG. 12).

[0148] Proliferation: Proliferation assays were performed for both cell lines, HeCaT cells and fibroblasts, at a protein coating concentration of 64 μl (i.e., 1:1 dilution in PBS) since no improved attachment was observed at higher coating concentrations in the previous assays. Briefly, 24-well plates were left to be coated overnight at 2-8°C, then washed once with PBS and subsequently air-dried under a BSC. Wells were seeded with 50,000 cells in a final total volume of 1 ml. All groups were performed in triplicate and assessed by OD measurements at 570 nm wavelength at 24, 48, and 72 hour intervals.

[0149] Proliferation assays showed enhanced proliferation for both fibroblasts and HeCaT cells, similar to previous studies (see FIG. 13). Nevertheless, the effect appears to be attenuated under the conditions tested here.

[0150] Migration: Migration assays were performed in non-culture treated 6-well plates with ibidi® inserts. Wells were coated overnight at 2-8°C with the same concentrations and conditions as in the proliferation assay. Similarly, wells were washed once with PBS and air-dried under a BSC. After complete drying, wells were equipped with ibidi® inserts as per the manufacturer's instructions. Each side of the ibidi® insert was then seeded with 20,000 cells per side to ensure cell confluency within the chambers of the insert. Cells were allowed to attach and grow for 24 hours before removing the ibidi® inserts. Immediately after removal of the ibidi® inserts, images were taken as a baseline measurement, and subsequently at three imaging time points (11:00AM, 1:00PM, and 3:00PM). Images were processed using Fiji (ImageJ) and their intensity values ​​were measured across the empty space left by removing the ibidi® wells. The gap was measured periodically over the three time points and closure was calculated.

[0151] Migration assays demonstrated a more pronounced effect on both cell types compared to previous studies (Figure 14). The most significant improvement is observed specifically for fibroblasts when compared to previous decellularization methods.

[0152] Results and Conclusions: The results suggest sufficient decellularization by NLS and mild dehydration, with DNA amounts of 24.29±0.007ng / ml (naive), 1.524±0.105ng / ml (NLS_TLC), and 1.772±0.186ng / ml (middle). Similar to the previous method (Example 3), the amount of DNA in the decellularized ECM is less than 10% of the DNA in the naive sample. Furthermore, the dried sample yielded 1.629±0.44ng / ml (translating to 17.15 nanograms of DNA per milligram of dried tissue). Based on cell attachment, proliferation, and migration assays, quantifiable protein bioactivity was retained after the decellularization and dehydration process.

[0153] One difference between the previous method (Example 3) and this new method is the tissue sample itself, which yielded significantly less DNA material (about 25 ng / ml) than the previous naive sample (about 100 ng / ml). This difference is supported by the results of the H&E analysis, which showed visibly fewer cells and other anatomical features than those found in the literature (Figures 9A-C). Furthermore, such differences may have affected the bioassay by attenuating the enhanced effect of ECM proteins on fibroblasts and HeCaT cells compared to the previous process. Additionally, while the previous method showed a more pronounced effect on HeCaT cells, this process appears to favor fibroblasts, especially when migration data is examined. In brief, migration data for the previous decellularization and dehydration protocol appears to favor HeCaT cells over fibroblasts. The results seem to suggest that this improved method of decellularization favors fibroblasts, but both types of cells can be used to demonstrate the functional bioactivity (proliferation and migration) of the decellularized ECM. Both keratinocytes (HeCaT cells) and fibroblasts play a role in the healing process.

[0154] Example 7 Treatment of subjects with gelatinized ECM Example 7.1 Topical serums for treating cuts A topical serum formulation is prepared containing 0.001%-5.0% weight / volume of gelatinized ECM and one or more carriers. The topical serum is applied twice daily, morning and night, to the site of the incision on the subject. At 8 weeks, the appearance of skin quality is observed.

[0155] Example 7.2 Topical treatment of skin abrasions The formulation is prepared to contain 0.001% to 5.0% weight / volume gelatinized ECM in a petrolatum base and, optionally, one or more other carriers. The formulation is placed on a non-stick gauze bandage. The bandage is applied to the subject's abrasion site. The bandage is changed daily and the abrasion site is observed to determine the number of days it takes for the skin to re-epithelialize.

[0156] For comparison, subject abrasions are treated with petrolatum alone. The abrasion sites are observed to determine the number of days it takes for the skin to re-epithelialize.

[0157] Example 7.3 Local treatment following an ablative laser resurfacing procedure The formulation is prepared to contain 0.001%-5.0% weight / volume of gelatinized ECM and one or more carriers or excipients. The formulation is applied daily to the skin of a subject after undergoing an ablative laser resurfacing procedure. The skin is observed daily for reduced inflammation and faster healing times.

[0158] Example 7.4 Topical treatments for treating burns The formulation is prepared to contain 0.001%-5.0% weight / volume gelatinized ECM in a petrolatum base and, optionally, one or more other carriers. A subject exhibiting a burn on the body is treated with the formulation. The formulation is placed on a dressing and applied to the site of the burn on the subject. The burn is checked daily and the dressing is changed daily or every 48 hours.

[0159] Example 8 Treatment of animal wounds with gelatinized ECM The objective of this study was to determine the ability of the ECM (test article) to enhance scar healing using a porcine third degree wound model and, in addition, to determine the ability of the ECM to enhance scar healing using a porcine 20 mm full thickness wound model.

[0160] Materials and Methods Experimental animals. A porcine model is used in this study due to the morphological similarities between porcine and human skin (Sullivan et al., 2001). Specific pathogen-free female animals (pigs) weighing 40-45 kg (BG Looper Farm, Inc., 4673 Petra Mill Road, Granite Falls, NC 28630) are kept in the facility for at least 5 days before the start of the experiment to allow the animals to acclimate. Animals are fed a basal diet ad libitum and housed individually in an animal facility (meeting the accredited American Association for Accreditation of Laboratory Animal Care [AAALAC]) with controlled temperature (19-21°C) and lighting (12 h / 12 ​​h light / dark).

[0161] Test Articles. Gelatinized ECM is prepared as described above.

[0162] Wound Technique. Twenty-seven third-degree burns are created on the paravertebral and thoracic regions. The burns are created by using a branding iron (L & H Manufacturing Company Mandan, North Dakota 58554) with a heat controller set at 300°C. The branding iron is held in a vertical position on the skin for 15 seconds with pressure provided by gravity, resulting in a burn 27 mm in diameter and approximately 3 mm deep (to the subcutaneous tissue). The wounds are separated from each other by 5-7 cm of unwounded skin. As seen in Experimental Schematic (ES) I, the wounds are randomly assigned to the three treatment groups with nine wounds per treatment. Twenty-seven full-thickness wounds are created with a 20 mm circular biopsy punch on the paravertebral and thoracic regions (see ES 1 below). The wounds are separated from each other by 5-7 cm of unwounded skin. Wounds are randomly assigned to three treatment groups with nine wounds per treatment as shown in ES 1. Animals are treated within 20 minutes of creation.

[0163] Experimental Scheme (ES) 1

[0164] [ka]

[0165] Treatment Regimen: Immediately after wounding, the wounds are treated with approximately 1000 ul of test article to cover the wound site and surrounding unwounded skin. After wound treatment, the wounds are covered with a polyurethane film dressing (Tegaderm; 3M Co., St. Paul, Minn.). All wounds are treated daily for 7 days. After 7 days, the wounds are covered with non-adherent gauze. All dressings are secured in place with tape and covered with Coban wrap (3M Co., St. Paul, Minn.).

[0166] Clinical Observations: Wounds from each group are photographed and the wound area is traced to measure wound contraction.

[0167] Digital Photography and Measurement of Wound Contraction. Wound perimeters are traced by digital imaging in ImageJ and compared to day 0 to determine the degree of wound contraction.

[0168] Erythema Measurements. During each evaluation time point, the amount of erythema (redness) around the wound is also scored clinically. Erythema scoring indicates the amount of inflammation present. Scoring is on a scale from 1 to 4 as follows: a score of 1 indicates an absence of inflammation; a score of 2 indicates mild inflammation; a score of 3 indicates moderate inflammation; a score of 4 indicates marked inflammation; and a score of 5 indicates severe inflammation.

[0169] Histological evaluation. Three incisional biopsies are taken from each treatment group using a sterile scalpel at the same evaluation time points, days 7, 14, and 45. Biopsies are taken through the center of the wound, including normal adjacent skin on both sides (see Experimental Scheme (ES) 2).

[0170] Experimental Scheme (ES) 2: Biopsy

[0171] [ka]

[0172] These specimens are placed in formalin and then stained with hematoxylin and eosin (H&E). One section per block is analyzed. The specimens are evaluated by light microscopy by a trained dermatopathologist blinded to their identity and examined as described below to determine treatment response.

[0173] At days 7, 12, 14, and 45, specimens are evaluated for percent of wound epithelialized, epithelial thickness, leukocyte infiltration, and granulation tissue formation.

[0174] Percentage (%) of wound epithelialized: The length of the wound surface covered with epithelium is measured.

[0175] Epithelial thickness (cell layers μm). Epithelial thickness may vary depending on the area within the biopsy. Epithelial thickness in μm is measured at five equally spaced points in the biopsy and averaged.

[0176] Leukocyte infiltration. Leukocyte infiltration is measured by the presence and amount of subepithelial mixed leukocyte infiltrate. Scoring is based on a mean score on a scale of 1 to 5, as follows: a mean score of 1 indicates absence of infiltration; a mean score of 2 indicates mild infiltration; a mean score of 3 indicates moderate infiltration; a mean score of 4 indicates marked infiltration; and a mean score of 5 indicates intense infiltration.

[0177] Granulation tissue formation. The approximate amount of new granulation tissue formation (dermis) is graded as follows: 0 is 0%; 0.5 is 1-10%; 1 is 11-30%; 2 is 31-50%; 3 is 51-70%; 4 is 71-90%; and 5 is 91-100%.

[0178] On day 45, the specimens are evaluated for scar formation and the dermis of the specimens is evaluated.

[0179] Scarring. Scarring is scored in the later stages of third-degree burns with Masson's Trichrome staining using a variety of criteria.

[0180] Epidermis: Scarring in the epidermis is graded looking for recovery of the epidermal ridges as follows: 0 is normal (normal recovery); 1 is thin (partial recovery); and 2 is thick (no partial recovery of the epidermal ridges).

[0181] Dermis: Collagen fiber orientation: 0 is normal (basket-weave); 1 is abnormal (<25%); 2 is abnormal (25-50%); 3 is abnormal (51-75%); and 4 is abnormal (76-100%). Density: the visual feature is high or low density, which is also scored as follows: 0 is normal (fasciculation density); 1 is abnormal (<25%); 2 is abnormal (25-50%); 3 is abnormal (51-75%); and 4 is abnormal (76-100%). Maturity: Visual characteristics are assessed (longer, shorter, thicker, or thinner). Maturity for each lesion is scored as follows: 0 is normal; 1 is abnormal (<25%); 2 is abnormal (25-50%); 3 is abnormal (51-75%); and 4 is abnormal (76-100%). Vascularity: A visual feature of high or low density, which is also scored as follows: 0 is normal; 1 is abnormal (<25%); 2 is abnormal (25-50%); 3 is abnormal (51-75%); and 4 is abnormal (76-100%). Thickness: 0 is normal; 1=thin; and 2=thick.

[0182] Molecular Evaluation. Also on the same evaluation day, additional 4 mm punch biopsies are taken from each wound (ES 2 above). Biopsies are immediately immersed in RNAlater stabilizing solution and incubated overnight at 4°C or frozen. Samples are then analyzed.

[0183] Immunohistochemistry evaluation. On the same evaluation day, three other incisional biopsies (see ES 2) are taken from the same wounds. The samples are then frozen and later analyzed.

[0184] All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, those skilled in the art will recognize that various modifications, substitutions, omissions, and changes can be made without departing from the spirit thereof. (References) Sullivan TP, Eaglstein WH, Davis SC, and Mertz PM. The pig as a model for human wound healing. Wound Repair and Regeneration 9, 2, 2001, 66-76 U.S. Pat. No. 1,061,7790

Claims

1. a gelatinized ECM comprising isolated amphibian ECM, wherein the isolated amphibian ECM has been decellularized; Contains intact native ECM structure; containing only 1%–10% of the total amount of DNA present in the corresponding native ECM sample; Contains residual DNA fragments of less than 300 base pairs (bp); and / or Gelatinized ECM, from which more than 70% of the cells have been removed.

2. the amphibian ECM contains 2% to 10% of the total amount of DNA present in the corresponding native ECM sample; or The amphibian ECM contains residual DNA fragments between 50 and 300 bp in length; or the ECM contains residual DNA fragments less than 250 bp in length; or Amphibian ECM is stripped of 80% to 90% of its cells. The gelatinized ECM of claim 1.

3. 2. The gelatinized ECM of claim 1, wherein the ECM is obtained from a juvenile amphibian, and optionally the juvenile amphibian comprises a froglet, tadpole, urodela, or juvenile Apoda.

4. The gelatinized ECM of claim 1, wherein the ECM is obtained from a juvenile urodele.

5. A composition comprising the gelatinized ECM of claim 1 and a carrier.

6. 6. The composition of claim 5, wherein the composition is a pharmaceutical or cosmetic composition, optionally wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier or the cosmetic composition comprises a cosmetically acceptable carrier.

7. 7. The composition of claim 6, wherein the composition further comprises one or more agents, optionally wherein the one or more agents are heterologous to the amphibian ECM.

8. further comprising one or more agents that are peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or combinations thereof; or further comprising one or more growth factors, cytokines, chemokines, or combinations thereof; or further comprising one or more polymers, optionally wherein said one or more polymers are synthetic or natural polymers or copolymers; 7. The composition of claim 6.

9. 1. A method for decellularizing amphibian ECM, comprising the steps of: collecting a biological sample from an amphibian; washing the sample with a detergent solution containing an anionic surfactant; treating the sample with one or more proteases and one or more nucleases to obtain isolated ECM; and dehydrating the isolated ECM under mild conditions.

10. 10. The method of claim 9, comprising the steps of: collecting a biological sample from an amphibian; rinsing the sample with a solution; washing the sample with a detergent solution comprising an anionic surfactant; treating the sample with a protease; rinsing the sample after the protease treatment; and treating the rinsed, protease-treated sample with a nuclease to obtain isolated ECM; and optionally dehydrating the isolated ECM under mild conditions; and further washing the isolated ECM before dehydrating.

11. 10. The method of claim 9, comprising rinsing with water or a buffer solution, optionally wherein the buffer solution is phosphate buffered saline (PBS) or Dulbecco's phosphate buffered saline (DPBS).

12. 10. The method of claim 9, comprising washing the sample with a detergent solution comprising N-lauryl sarcosine (NLS), potassium lauryl sarcosine, cholate, deoxycholate, sodium dodecyl sulfate (SDS), or polyoxyethylene sorbitan fatty acid ester, or Tween, optionally wherein the Tween is Tween 20, 40, 60, or 80.

13. The method of claim 1, wherein treating the sample with one or more proteases comprises incubating the sample with one or more proteases, including one or more aminoendopeptidases, and optionally the one or more proteases comprise dispase I, dispase II, trypsin, papain, or collagenase; and / or treating the sample with one or more nucleases comprises incubating the sample with one or more nucleases comprising a non-specific DNA / RNA endonuclease, and optionally the one or more nucleases comprise a benzonuclease, a turbonuclease, a pulmozyme, or a combination of a deoxyribonuclease and a ribonuclease; The method of claim 9.

14. The following steps are performed in the following order: collecting a biological sample from the amphibian; rinsing the sample with buffered saline; washing the sample with a detergent solution containing NLS; rinsing the sample with buffered saline; treating the rinsed sample with Dispase II; rinsing the sample with buffered saline; and treating the rinsed sample with benzonuclease to obtain isolated ECM; and Optionally, dehydrating the sample under mild conditions and / or washing the isolated ECM with DPBS prior to dehydrating.

10. The method of claim 9, comprising:

15. 11. The method of claim 10, wherein the step of dehydrating under mild conditions comprises dehydrating under a reduced pressure of 360 Torr at 15°C.

16. 10. The method of claim 9, wherein the sample is washed with the detergent solution for 6 hours with shaking.

17. 10. The method of claim 9, wherein the sample is rinsed with DPBS for 5 minutes between the detergent solution washing step and the protease treatment step, and between the protease treatment step and the nuclease treatment step.

18. The biological sample is derived from a froglet, tadpole, urodele, or juvenile larval stage of Apoda; or the biological sample comprises ECM tissue from a juvenile urodele; or The biological sample comprises ECM of connective tissue, adipose tissue, bone, plasma, skin, cartilage, tendon, dura mater, or fascia; The method of claim 9.

19. A method for gelatinizing isolated ECM obtained by the method of claim 9, comprising the step of dissolving the isolated ECM in a solution containing a weak acid and an endopeptidase to form gelatinized ECM.

20. A pharmaceutical composition comprising the gelatinized ECM of claim 1, for the treatment and / or prevention of skin conditions including fine lines and / or wrinkles, aging, redness, abrasions, burns, cuts, infections, razor burn, scars, uneven skin tone, soreness, stretch marks, increasing skin elasticity and / or firmness, improving skin hydration, inflammation, and hyperpigmentation; or To protect the skin from damage caused by UV rays and / or environmental pollution Pharmaceutical compositions.

Images