Activated amphibian cells and uses thereof

Abstract

This disclosure describes compositions comprising activated amphibian bud bases, which can be used to treat various skin diseases and conditions. Bud bases harvested from the injured site of an amphibian 1 to 7 days after injury and subsequently activated are effective in treating skin diseases and conditions.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 602,257, filed November 22, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure describes compositions comprising activated amphibian budding cells and their use in wound treatment. Background Technology

[0003] As the largest organ in the mammalian body, the skin is the body's outer covering, serving three main functions: preventing microorganisms and excessive water loss, regulating body temperature, and providing sensations of touch, heat, and cold. Mammalian skin has two main layers: the epidermis and the dermis. The epidermis, the outermost layer of skin that prevents microorganisms from entering the body and retains moisture, is a stratified squamous epithelium composed of keratinocytes. The dermis is the layer of skin beneath the epidermis. As a skin appendage, the dermis provides elasticity through an extracellular matrix composed of collagen fibers, elastic fibers, hyaluronic acid, and proteoglycans. The dermis and epidermis are separated by a thin sheet of fibers called the basement membrane, which regulates the flow of cells and molecules (such as cytokines and growth factors) between the dermis and epidermis during remodeling, repair, and regeneration. Below the dermis lies the subcutaneous tissue, composed of loose connective tissue (such as fat and elastin). The main cells of the subcutaneous tissue include fibroblasts, macrophages, and adipocytes.

[0004] Injury or disease can disrupt the morphology and function of organs or tissues, such as the skin. Post-injury remodeling and repair of organs or tissues is a complex wound healing process involving interactions between cells, growth factors, and the extracellular matrix (ECM). In adult mammals, this process involves well-known phases: homeostasis, inflammation, proliferation, maturation, and remodeling. During homeostasis, coagulation occurs to stop bleeding. Inflammation involves recruiting leukocytes, antibodies, nutrients, and enzymes to the affected area to accelerate wound healing. During proliferation, new healthy granulation tissue, such as new connective tissue and blood vessels, replaces the wound. Maturation and remodeling occur after wound closure and involve the repair of dermal tissue to increase its tensile strength.

[0005] In contrast to repair processes that aim to restore function without considering the exact location of damaged tissue, regeneration replaces injured tissue with an exact copy, resulting in a complete restoration of both morphology and function. For example, uninjured skin continuously and completely regenerates as new cells are replaced. However, injured skin in adult mammals does not fully regenerate and leaves scars after healing.

[0006] While all mammals, including humans, can spontaneously regenerate their fingertips after adulthood, mammals cannot regenerate their limbs, unlike amphibians. Most amphibians can regenerate missing body parts. However, their regenerative abilities vary greatly between species. For example, tailed animals have excellent regenerative abilities.

[0007] In amphibians, in response to amputation, cells from the surrounding limb tissue dedifferentiate and produce a large number of undifferentiated and proliferating cells called budding cells, which are capable of regenerating into body parts. Some amphibians can produce budding cells even in adulthood, enabling them to regenerate even in adulthood. The embryonic bud plays a crucial role in the regeneration process.

[0008] Skin wound repair in adult mammals typically results in scar tissue formation. Unlike mammals, amphibians repair wounds through regeneration rather than scarring. The goal is to develop methods that do not cause scarring and can treat mammalian wounds quickly. Summary of the Invention

[0009] This summary is provided to present, in a simplified form, the selection of concepts that will be further described in the detailed embodiments below. This summary is not intended to identify all key or essential features of the claimed subject matter, nor is it intended to be used alone to help determine the scope of the claimed subject matter.

[0010] This disclosure provides compositions comprising activated bud bases. The bud bases are obtained from amphibians less than 7 days after injury. In embodiments, the amphibians are juvenile caudal animals, such as axolotls.

[0011] This disclosure also provides a method for preparing activated bud bases, the method comprising: injuring an amphibian; allowing the amphibian to recover from the injury; isolating the bud base from the injury site of the amphibian less than 7 days after the injury; and activating the isolated bud base to induce the release of intracellular components from the bud base cells. Activation of the isolated bud base includes mechanical or chemical means, including homogenization, use of a detergent, or cell lysis to lyse the bud base.

[0012] This disclosure also provides the use of activated bud bases to treat or prevent skin diseases, conditions, or wounds in subjects. Attached Figure Description

[0013] Figure 1Experimental design for animals with third-degree burns. Following injury (third-degree burns), Z gel, T gel, Y gel, F gel, or X gel were applied to various sites on the animals, respectively. H was the untreated control group. Z gel (PowerBlast day 1) consisted of activated buds harvested one day after amphibian injury and applied to wounds #3, #12, and #13. T gel (PowerBlast day 2) consisted of activated buds harvested two days after amphibian injury and applied to wounds #4, #5, and #14. Y gel (PowerBlast day 3) consisted of activated buds harvested three days after amphibian injury and applied to wounds #8, #9, and #22. F gel (PowerBlast day 4) consisted of activated buds harvested four days after amphibian injury and applied to wounds #18, #21, and #23. X-gel (PowerBlast 7 days) consisted of activated buds harvested 7 days post-injury in amphibians and applied to wounds #1, #7, and #19. Wounds #10, #15, and #24 served as untreated control groups (H). All wounds were treated for 7 days (from day 0 of the initial trauma until day 6). Following treatment application, all wounds were covered with a polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) until day 11, after which the Tegaderm dressing was replaced with non-adhesive gauze until the final assessment day (day 14 post-traumatic injury). In each treatment group, two wounds were treated with topical application, and a third wound (strip) underwent laser-assisted delivery (LAD). Wounds #5, #9, #12, #15, #18, and #19 were laser-treated.

[0014] Figure 2Experimental design in animals with deep dermal wounds. Following injury (deep dermal injury), Z gel, T gel, Y gel, F gel, or X gel were applied to various sites on the animals. H was the untreated control group. Z gel consisted of activated buds harvested one day after amphibian injury and applied to wounds #3, #14, and #12. T gel consisted of activated buds harvested two days after amphibian injury and applied to wounds #4, #15, and #25. Y gel consisted of activated buds harvested three days after amphibian injury and applied to wounds #8, #9, and #20. F gel consisted of activated buds harvested four days after amphibian injury and applied to wounds #5, #19, and #22. X-gel, consisting of activated budding material harvested seven days post-injury in amphibians, was applied to wounds #1, #7, and #16. Wounds #10, #21, and #24 served as untreated control groups (H). All wounds were treated for seven days (from day 0 of the initial trauma until day 6). Following treatment application, all wounds were covered with a polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) until the final assessment day (day 10 post-traumatic). In each treatment group, two wounds were treated with topical application, and the third wound (strip) underwent laser-assisted delivery (LAD). Wounds #1, #4, #9, #14, #22, and #24 were laser-treated.

[0015] Figure 3 Experimental design for animals with 20 mm full-thickness wounds. Following injury (deep dermal injury), X gel, Y gel, or Z gel was applied to various sites on the animals. H was the untreated control group. X gel consisted of activated budding material harvested seven days after amphibian injury and applied to wounds #1, #7, and #23. Y gel consisted of activated budding material harvested three days after amphibian injury and applied to wounds #8, #9, and #18. Z gel consisted of activated budding material harvested one day after amphibian injury and applied to wounds #3, #13, and #24. Wounds 12, 19, and 22 served as the untreated control group (H). In each treatment group, two wounds were treated with topical application, and the third wound (circled) underwent laser-assisted delivery (LAD). Wounds #13, #18, #19, and #23 were laser-treated.

[0016] Figures 4A1 to 4C Percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds. Different wounds from Test B and Test A were treated with Z gel (one day). Figure 4A1 and Figure 4A2For Experiment B and Experiment A, the percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 4B For Experiment B, the percentage of epithelialization in deep dermal wounds was determined with and without laser pretreatment. Figure 4C For Experiment A, the percentage of epithelialization of full-thickness wounds with and without laser pretreatment.

[0017] Figures 5A to 5B Percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds. Different wounds in Experiment B were treated with T gel (for two days). Figure 5A For Experiment B, the percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 5B For Experiment B, the percentage of epithelialization in deep dermal wounds with and without laser pretreatment.

[0018] Figures 6A1 to 6C Percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds. Different wounds from Test B and Test A were treated with Y gel (for three days). Figure 6A1 and Figure 6A2 For Experiment B and Experiment A, the percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 6B For Experiment B, the percentage of epithelialization in deep dermal wounds was determined with and without laser pretreatment. Figure 6C For Experiment A, the percentage of epithelialization of full-thickness wounds with and without laser pretreatment.

[0019] Figures 7A1 to 7B2 Percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds. Different wounds in Test B were treated with F gel (for four days). Figure 7A1 and Figure 7A2 For Experiment B, the percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 7B1 and Figure 7B2 For Experiment B, the percentage of epithelialization in deep dermal wounds with and without laser pretreatment.

[0020] Figures 8A1 to 8C Percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds. Different wounds from Test B and Test A were treated with X gel (for seven days). Figure 8A1 and Figure 8A2 For Experiment B and Experiment A, the percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 8B For Experiment B, the percentage of epithelialization in deep dermal wounds was determined with and without laser pretreatment. Figure 8C For Experiment A, the percentage of epithelialization of full-thickness wounds with and without laser pretreatment.

[0021] Figures 9A1 to 9B2 The mean percentage of epithelialization (%) in third-degree burn wounds, deep dermal wounds, and full-thickness wounds from Experiment B. Figure 9A1 and Figure 9A2 For Experiment B, the mean percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 9B1 and Figure 9B2 For Experiment B, the average percentage of epithelialization of deep dermal wounds with and without laser pretreatment.

[0022] Figures 10A1 to 10B2 The mean percentage of epithelialization (%) in third-degree burn wounds and full-thickness burns from Experiment A. Figure 10A1 and Figure 10A2 For Experiment A, the mean percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 10B1 and Figure 10B2 For Experiment A, the average percentage of epithelialization of the full-thickness wound with and without laser pretreatment.

[0023] Figures 11A to 11D Percentage of epithelialization (%) in third-degree burn wounds and deep dermal wounds in Experiment B. Experiment B used X gel, F gel, Y gel, T gel, and Z gel to treat third-degree burn wounds and deep dermal windows. Figure 11A For Experiment B, the percentage of epithelialization in third-degree burn wounds without laser pretreatment. Figure 11B For Experiment B, the mean percentage of epithelialization in third-degree burn wounds without laser pretreatment. Figure 11C For Experiment B, the percentage of deep dermal wound epithelialization without laser pretreatment. Figure 11D For Experiment B, the average percentage of epithelialization in deep dermal wounds without laser pretreatment. Figures 12A1 to 12B2 The mean percentage (%) of epithelialization in third-degree burn wounds, deep dermal wounds, and full-thickness wounds in Experiments A and B. Figure 12A1 and Figure 12A2 For Experiment A and Experiment B, the mean percentage of epithelialization in third-degree burn wounds with and without laser pretreatment. Figure 12B1 and Figure 12B2For Experiment A and Experiment B, the average percentage of epithelialization in deep dermal wounds and full-thickness wounds with and without laser pretreatment.

[0024] Figure 13 Epithelial thickness of a third-degree burn wound on day 14 post-injury without laser treatment.

[0025] Figure 14 Epithelial thickness of a third-degree burn wound on day 14 post-injury when laser treatment is used.

[0026] Figure 15 Leukocyte infiltration in a third-degree burn wound on day 14 post-injury without laser treatment.

[0027] Figure 16 Leukocyte infiltration in a third-degree burn wound 14 days post-injury when laser treatment was used.

[0028] Figure 17 Granulation tissue of a third-degree burn wound on day 14 post-injury without laser treatment.

[0029] Figure 18 Granulation tissue of a third-degree burn wound 14 days after injury, when treated with laser.

[0030] Figure 19 Percentage of re-epithelialization of deep dermal wounds on day 10 post-injury without laser treatment.

[0031] Figure 20 The percentage of deep dermal wounds re-epithelialized on day 10 post-laser treatment.

[0032] Figure 21 Epithelial thickness of deep dermal wounds on day 10 post-injury without laser treatment.

[0033] Figure 22 Epithelial thickness of deep dermal wounds on day 10 post-laser treatment.

[0034] Figure 23 Leukocyte infiltration in deep dermal wounds on day 10 post-injury without laser treatment.

[0035] Figure 24 Leukocyte infiltration in deep dermal wounds on day 10 post-injury when laser treatment is used.

[0036] Figure 25Granulation tissue formation in deep dermal wounds on day 10 post-injury without laser treatment.

[0037] Figure 26 Granulation tissue formation in deep dermal wounds on day 10 post-injury when laser treatment is used.

[0038] Figure 27A and Figure 27B The wound area of ​​treated third-degree burns decreased over time. Figure 27A Wound area measurements were taken from day 0 to day 14. Figure 27B Measurement of third-degree burn wounds under laser treatment.

[0039] Figure 28 Measurement of third-degree burn wounds without laser treatment.

[0040] Figure 29 Measurement of deep dermal wounds when laser treatment is used.

[0041] Figure 30 Measurement of deep dermal wounds without laser treatment. Detailed Implementation

[0042] Unlike mammals, amphibians such as Urodele regenerate their skin structures (including the dermis and glands) without scarring after deep skin injuries. Demircan reported that amphibian tissue could heal wounds in mice. Demircan only disclosed the use of amphibian bud-based tissue grafts or bud-based cells on wounds. Demircan described the bud-based stage of bud-based application between 24 hours and 4 weeks, and preferably on day 7, 10, or 15 post-amputation (WO 2020034902).

[0043] Therefore, it has been surprisingly found that bud bases harvested from the injury site of amphibians less than 7 days after injury and subsequently activated possess wound healing potential. In an embodiment, activated bud bases prepared from amphibian bud bases harvested 12 hours to 6 days, 1 to 5 days, 2 to 4 days, 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days after injury possess wound healing potential. In an embodiment, bud base cells can be activated 1 to 5 days, 2 to 4 days, or 3 days after injury to have enhanced wound healing potential.

[0044] The terms “a,” “an,” “the,” and similar indicators used in the context of describing the invention (particularly in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by the context.

[0045] The term "activated bud base" refers to a bud base harvested from an amphibian a certain number of days after injury and subsequently activated to induce the release of intracellular components from the bud base cells.

[0046] The term "dermatitis" includes skin conditions that require treatment with "medications," including diseases, defects, and injuries that include wounds, such as burns, deep dermal wounds, and full-thickness wounds.

[0047] The term "cosmetic dermatitis" includes dermatitis related to skin tone, clarity, luster, brightness, and / or hydration.

[0048] The term "subject" refers to an animal, such as a mammal. Examples of mammals include humans, dogs, cats, horses, cattle, goats, sheep, pigs, or non-human primates. Subjects requiring treatment or having this need include subjects suffering from a disease or condition requiring treatment. Subjects having this need include subjects requiring treatment and / or prevention of skin conditions or subjects with wounds requiring treatment.

[0049] The term "therapeutic effective amount" refers to the amount of a product or composition (e.g., a pharmaceutical product) that provides therapeutic benefit in treating, preventing, or controlling a disease or condition such as a skin disease, skin injury, or wound. The term "therapeutic effective amount" also includes the amount of one or more compounds that, when applied, are sufficient to prevent the development of one or more signs or symptoms of the treated disorder or disease, or to alleviate, to some extent, the signs or symptoms of the treated disorder or disease.

[0050] In the context of administering a product, such as a biomaterial, to a subject, the term "treatment" or "manipulation" refers to the application of a product to achieve a desired clinical / medical endpoint, including the relief of symptoms of a disease or condition. Examples of such desired endpoints related to skin diseases or conditions include wound healing, tissue closure, tissue expansion, prevention of tissue adhesion, provision of structural support to tissues, provision of a protective barrier, and / or correction of defects. Administering a product also includes applying the product to the subject.

[0051] The term "heterogeneous" refers to a product that is derived from or originates from a member of another species.

[0052] The term "amphibians" refers to cold-blooded vertebrates, including frogs, toads, salamanders, and fire salamanders. They have an aquatic gill-breathing larval stage, followed by a terrestrial lung-breathing adult stage. Amphibians include the class Amphibia and the orders Anura (frogs or toads), Caudata (salamanders or fire salamanders), and Apoda (caecilians). In embodiments, the amphibians described herein are larval or neotenous amphibians. Larval amphibians include larval frogs, such as young frogs, tadpoles, or larval Apoda. Amphibians can be larval amphibians, and larval amphibians can include the larval stage of any order of amphibian. Amphibians can be necrotened.

[0053] The term "salamander" refers to a group of amphibians characterized by their lizard-like appearance and the fact that they have a tail throughout their lives. Salamander families include: Ambystomatidae (mole salamander), Amphiumidae (Congo eel), Cryptobranchidae (giant salamander), Dicamptodontidae (Pacific giant salamander), Hynobiidae (Asiatic salamander), Plethodontidae (lungless salamander), Proteidae (mudpuppie and olm), and Rhyacotritonidae (torrent salamander). Salamanders (Salamandridae, newts and salamanders) and Sirenidae (Sirens).The family Ambystoma includes the following: *Ambystoma altamirani*, *Ambystoma amblycephalum*, *Ambystoma andersoni*, *Ambystoma annulatum*, *Ambystoma barbourin*, *Ambystoma bishop*, *Ambystomabombypellum*, *Ambystoma californiense*, *Ambystoma cingulated*, *Ambystoma dumerilii*, *Ambystoma flavipiperatum*, and *Ambystoma fibrous*. The following are a list of salamander species: *Ambystoma gracile*, *Ambystoma granulosum*, *Ambystoma jeffersonianum*, *Ambystoma laterale*, *Ambystoma leorae*, *Ambystoma lermaense*, *Ambystoma mabeei*, *Ambystoma macrodactylum*, *Ambystoma maculatum*, *Ambystoma mavortium*, *Ambystoma mexicanum*, *Ambystoma opacum*, *Ambystoma ordinarium*, *Ambystoma rivulare*, and *Ambystoma rubigina*. The species include *Ambystoma rosaceum*, *Ambystoma silvense*, *Ambystoma subsalsum*, *Ambystoma talpoideum*, *Ambystoma taylori*, *Ambystomatexanum*, *Ambystoma tigrinum*, and *Ambystoma velasci*.

[0054] Salamanders are classified under the order Urodela (or Caudata). The term "Urodela" refers specifically to salamanders, which belong to the order Urodela within the class Amphibia. Salamanders can originate from both Urodela and Anea. Urodela begin their lives as aquatic larvae, and some undergo metamorphosis from a gill-bearing larval stage to a lung-bearing, air-breathing adult stage. During metamorphosis, the physical characteristics of Urodela change to prepare them for terrestrial life. These changes include tail fin absorption, thickening of the skin, development of dermal glands, and gill absorption. Sexual maturity in most Urodela also occurs during this period. However, some families of Urodela are "neocontinental," meaning that individuals in these families retain their larval aquatic form throughout their lives, even after reaching sexual maturity. The Mexican axolotl (Ambystoma mexicanum), the Mexican axolotl, and / or hybrids of the Mexican axolotl and the tiger axolotl are examples of juvenile axolotls. Adult Mexican axolotls do not become terrestrial amphibians, but remain aquatic and possess gills. However, under certain circumstances, Mexican axolotls undergo metamorphosis and transform into a terrestrial form.

[0055] Axolotls can completely regenerate missing or damaged body parts, including organs, limbs, and parts of the central nervous system, throughout their lives. Aquatic axolotls undergo rapid reepithelialization during wound healing and limb regeneration, both of which are scarless processes. Similarly, metamorphosed terrestrial axolotls retain several larval skin features and also exhibit scarless wound healing, but at a slower rate than pre-metamorphosed aquatic axolotls. The wound healing process in axolotls is similar to the scarless healing process of wounds in mammalian fetuses and embryos. This wound exhibits reepithelialization and basement membrane remodeling, which occur more rapidly than corresponding events in postnatal mammals.

[0056] A budding base is an undifferentiated cluster of cells that can develop into an organ. In amphibians, the budding base is crucial for limb regeneration. Budding bases are present in early stages of organism development (e.g., embryonic development) and in the regeneration of tissues, organs, and bones. Some amphibians (such as salamanders) produce budding bases even in adulthood, enabling them to regenerate their limbs, tails, and other organs after amputation. The axolotl is a juvenile salamander with remarkable regenerative abilities. Limb regeneration in these salamanders involves budding bases.

[0057] Spores isolated from amphibians (such as caudal animals) less than 7 days after injury and subsequently activated to induce the release of intracellular components from the spore base have wound healing potential. Spores can be harvested from the injury site at 1, 2, 3, 4, 5, or 6 days after injury and then activated for wound treatment. In this embodiment, spores harvested and subsequently activated at 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, or 3 days after injury have wound healing potential.

[0058] Damage to an amphibian in order to produce budding can include different body parts of the amphibian, such as the forelimbs, which include the forelegs, feet and four toes; the hindlimbs, which include the hind legs, feet and five toes; the tail; the spinal cord; the lower jaw; the brain, which includes the telencephalon or forebrain; the heart, which includes the ventricles; and part of the eye.

[0059] After harvesting the shoot stalk from the damaged site, the stalk can be activated through physical, chemical, and / or electrolytic disruption involving cell lysis to induce the release of intracellular components from the stalk. Examples of physical disruption methods include mechanical disruption using devices such as stirrers, homogenization using ultrasonic homogenizers, sonication, freeze-thaw cycles, and manual grinding. Chemical disruption involves lysing cells using anionic, nonionic, or zwitterionic detergents. Examples of such detergents include sodium lauryl sulfate (SDS), Triton X-100 (nonionic), Triton X-114 (nonionic), NP-40 (nonionic), Tween 20 (nonionic), Tween 80 (nonionic), CHAPS (zwitterionic), and CHAPSO (zwitterionic). Other forms of cell lysis include cell lysis and osmotic lysis. Electrolytic disruption involves applying a high electric field to the stalk to disrupt the cell membrane. An example of electrolytic disruption is irreversible electroporation.

[0060] The activated bud substrate described herein exhibits enhanced properties. For example, compared to control wound sites, activated bud substrate shows enhanced properties at the wound site. The control group can be untreated, for example, a wound site not treated with the activated bud substrate. The control group can also be a wound site treated with the activated bud substrate, which is prepared from bud substrate harvested more than 7 days after injury. The control group can also be a wound site treated with unactivated bud substrate.

[0061] This application describes compositions comprising activated amphibian bud bases. The activated bud base is a mixture of amphibian bud base cells that have been destroyed to release intracellular components from the bud base. The activated amphibian bud base can be obtained from various injured amphibians (such as caudal animals) described herein. In an embodiment, the amphibian bud base is obtained from an injured axolotl.

[0062] The compositions described herein may include carriers and / or excipients. Examples of carriers and excipients include brine, emulsions, mixtures of organic solvents and water, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, petrolatum, lanolin, mineral oil, polydimethylsiloxane, humectants, and polyethylene glycol. Examples of humectants include glycerin, lecithin, and propylene glycol. In embodiments, the compositions described herein include cosmetic compositions or pharmaceutical compositions, each comprising one or more cosmetically or pharmaceutically acceptable carriers or excipients.

[0063] The compositions described herein may include a carrier for use in an immediate or sustained-release formulation. Such a carrier includes a polymer. The polymer may be biodegradable and / or bioabsorbable. As an example, for controlled release, the biomaterial may be coated with a polymer, such as acrylic polymers, acrylic / methacrylic acid copolymers, cellulose acetate phthalate (CAP), or opalrylamide. ® And Ethocel™. For immediate release, the biomaterial can be coated with cellulose polymers such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), methylcellulose (MC), and sodium carboxymethyl cellulose (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.

[0064] The compositions described herein may include one or more agents, such as therapeutic agents or cosmetic agents. Examples of therapeutic agents include known pharmaceuticals, such as retinoic acid, corticosteroids, antifungals, antiviral agents, antibiotics, preservatives, local anesthetics, and antitumor drugs. Examples of cosmetic agents include antioxidants, peptides, alpha- or beta-hydroxy acids, retinol, vitamins, plant extracts, skin clearers such as arbutin, moisturizers such as hyaluronic acid, emollients, carbohydrates, glycoproteins, and / or polymers. One or more agents may include combinations of agents. For activated buds, the agent may be exogenous or heterogenous.

[0065] Examples of one or more peptides and proteins include growth factors, cytokines, and chemokines. Examples of growth factors include: fibroblast growth factor (FGF), 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 factor 1 and insulin-like growth factor 2 (IGF-1 and IGF-2); keratinocyte growth factor (KGF); nerve growth factor (NGF); neurotrophic factors, such as neurotrophic factor-3, neurotrophic factor-4, and neurotrophic factor-5; platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); and stromal-derived factor 1α. Examples of cytokines and chemokines include tumor necrosis factor-α (TNF-α), interleukin-1α and interleukin-1β (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.

[0066] Examples of 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 antimicrobial 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, fenprofen, sulindac, diflunisal, diclofenac, indoprofen, and sodium salicylate.

[0067] Examples of cosmetic reagents include one or more glycoproteins, including proteoglycans (proteins covalently linked to glycosaminoglycans (GAGs)), antioxidants, ascorbic acid, vitamin C, alpha-hydroxy acids (AHAs), beta-hydroxy acids (BHAs), exfoliants, skin brighteners, light diffusing agents, UV absorbers, sunscreens, moisturizers, anti-wrinkle ingredients, and oil-absorbing agents. Examples of AHAs include glycolic acid, lactic acid, malic acid, tartaric acid, and citric acid. Examples of BHAs include salicylic acid.

[0068] The compositions described herein may also include one or more natural and / or synthetic polymers. Natural polymers may be derived from animal sources or non-animal sources such as plant sources. Examples of natural polymers include natural polymers such as collagen, chitosan, alginate, glycosaminoglycans, fibrous proteins, 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), polyethylene glycol diacrylate (PEG diacrylate), polyhydroxy acids, polydioxanone, polycaprolactone, polyorthoesters, polyanhydrides, polyphosphazenes, polyamino acids, pseudo-polyamino acids, conductive polymers (e.g., polyacetylene, polypyrrole, polyaniline), polyurethane, polystyrene, and nickelitanol.

[0069] The polymers in the compositions described herein can be biocompatible, biodegradable, and / or bioabsorbable, and can be random copolymers, block copolymers, or blends of monomers, homopolymers, copolymers, and / or hybrids containing these monomers. Exemplary biodegradable or bioabsorbable polymers include polylactide, polyglycolic acid, polycaprolactone, polydioxane, and their random copolymers and block copolymers. Biodegradable and / or bioabsorbable polymers may contain monomers selected from the group consisting of glycolide, lactide, dioxane, caprolactone, trimethylene carbonate, ethylene glycol, and lysine. Biodegradable and / or bioabsorbable polymers may contain bioabsorbable and biodegradable linear aliphatic polyesters, such as polyglycolic acid (PGA) and its random copolymer poly(glycolic acid-co-lactide) (PGA-co-PLA). Other examples of suitable biocompatible polymers include polyhydroxyalkyl methacrylate, ethyl methacrylate, polyvinylpyrrolidone, and polyacrylamide. Other suitable bioabsorbable materials are biopolymers, including collagen, gelatin, alginate, chitin, chitosan, fibroin, hyaluronic acid, dextran, polyamino acids, polylysine, and copolymers of these materials. Any combination of the polymers and copolymers of the examples above, or blends thereof, may also be included in the composition.

[0070] The compositions described herein may also include protectants, adsorbents, modifiers, emollients, preservatives, antioxidants, humectants, buffers, solubilizers, skin penetration enhancers, and surfactants.

[0071] Skin penetration enhancers can be added to the compositions described herein, provided that the skin penetration enhancer is safe and can effectively promote the penetration of desired substances from the activated bud matrix across the skin membrane. Examples of skin penetration enhancers include dimethyl sulfoxide (DMSO), monoglycerides, C10 to C20 fatty acid esters (including ethyl palmitate and isopropyl myristate); acyl lactates, such as hexanoyl lactate and lauroyl lactate; dimethyl lauramide; dodecyl acetate; lactates, such as lauryl lactate and myristyl lactate; monoalkyl ethers of polyethylene glycol and their alkyl or aryl carboxylic acid esters and carboxymethyl ethers (e.g., polyethylene glycol-4 lauryl ether (lauryl alcohol polyether-4)). ) and polyethylene glycol-2 lauryl ether (lauryl ether-2); myristyl ether-3, myristoyl sarcosine and methyl laurate; polypropylene glycol, polyethylene glycol, lecithin, urea, amino acids, 1-dodecyl hexahydro-2H-azapyr-2-one (azone), oleic acid, linoleic acid, isopropyl linoleate, oleyl alcohol, 1-dodecyl-azapyrane-2-one, butanediol and diethylene glycol monoethyl ether (transcutol).

[0072] The compositions described herein may be in the form of dry powder, solution, paste, liquid, extract, cream, lotion, serum, emollient, ointment, dispersion, gel, hydrogel, gelatin or emulsion.

[0073] The compositions described herein can also be used to treat and / or prevent skin diseases and conditions. The compositions described herein can be used to treat skin diseases and conditions (including wounds) and enhance wound healing without leaving any scars. The compositions described herein can reduce inflammation, reduce scarring, reduce keloid formation, reduce or alleviate the severity of scar formation and keloid formation, and / or reduce healing time for various dermatological and cosmetic surgeries. The compositions described herein can also be used to restore lost dermal matrix or subcutaneous volume.

[0074] Skin diseases or conditions include inflammatory and cancerous skin conditions. Examples of skin conditions requiring treatment include acne, actinic keratosis, vesicles, cellulitis, cold herpes, urticaria, impetigo, keratosis pilaris, melasma, nevus, tinea, 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 chronic simple lichen simplex; conditions of hair follicles and sebaceous glands such as acne, rosacea and rhinophyma, perioral dermatitis and pseudofolliculitis beardii; 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 bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. Burns include acute thermal burns, such as first-degree, second-degree, or third-degree burns. Skin conditions also include wounds, including third-degree burns, deep dermal wounds, and full-thickness wounds.

[0075] The compositions described herein can be applied topically to target sites or by injection, implantation, microneedling, radiofrequency microneedling, or the use of ablative fractional lasers. The isolated compositions described herein can be administered to a subject before, during, or after a dermatological or cosmetic procedure, such as dermabrasion, microdermabrasion, or ablative laser resurfacing. Dermatological or cosmetic procedures include the removal of at least one cell from the stratum corneum. The compositions described herein can also be delivered as injectables or together with dermal or subcutaneous implants, such as volume fillers, hyaluronic acid, or other dermal matrix proteins, including naturally occurring, bioengineered, or recombinant collagen or elastin. The compositions described herein can be administered alone or in combination with one or more of the agents described herein, such as growth factors, peptides, and proteins. The compositions can also be administered together with toxins, such as botulinum toxin.

[0076] The compositions described herein can be applied topically, such as directly to a wound or skin, or indirectly by applying them to a base for covering a wound or skin. The compositions can also be applied to a device, such as a medical device, for administration to a subject.

[0077] Before treatment with activated budding material, the wound site can be treated with lasers, microdermabrasion, microneedling, or any dermatological or cosmetic procedure that removes at least one stratum corneum cell. These procedures can help deliver the activated budding material and promote wound healing without scarring. Laser treatment includes treating the wound with ablative fractional lasers. Microneedling includes treating the wound with radiofrequency microneedling.

[0078] In the embodiments described herein, the composition can increase the epithelial thickness of the wound, thereby providing strong skin at the wound site. The composition can also increase the re-epithelialization rate, indicating that it can accelerate the wound healing process. Furthermore, the composition exhibits wound closure efficiency, particularly for third-degree burns, as demonstrated by achieving 100% re-epithelialization by day 14 after laser treatment. Further, the composition can reduce leukocyte infiltration, indicating that it can minimize scarring and accelerate recovery.

[0079] This disclosure describes a kit comprising the compositions described herein for the various uses described herein. The kit may include sterile compositions of any shape and form. The kit may include solutions for reconstituted composition for use. The kit may include devices for administering the composition to a subject. The kit may include implants coated with the composition prior to implantation into a subject. The kit may include components for the various uses described herein.

[0080] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise obviously contradicted by the context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any unclaimed element necessary for the practice of the invention.

[0081] The figures used in the specification and claims to indicate ranges or quantities of ingredients, components, reaction conditions, etc., should be understood to be modified by the term "about". When further clarification is required, the term "about" has the meaning reasonably assigned by a person skilled in the art when used in conjunction with a specified value or range, namely, slightly larger or smaller than the specified value or range, within ±20% of the specified value; ±15% of the specified value; ±10% of the specified value; ±5% of the specified value; ±4% of the specified value; ±3% of the specified value; ±2% of the specified value; ±1% of the specified value; or any percentage between ±1% and 20% of the specified value.

[0082] As will be understood by those skilled in the art, each embodiment disclosed herein may include, consist substantially of, or be composed of the elements, steps, components, or components specifically stated herein. Therefore, the term “comprising” should be interpreted as: “comprising, consisting of, or substantially consisting of.” The transitional terms “comprising” or “including” mean, but are not limited to, and allow the inclusion of unspecified elements, steps, components, or components, even in large quantities. The transitional phrase “consisting of” excludes any unspecified elements, steps, components, or components. The transitional phrase “substantially consisting of” limits the scope of the embodiments to the specified elements, steps, components, or components and those that do not substantially affect the embodiment. In an embodiment, a lack of substantial effect is manifested as statistically significant wound healing as determined by one or more of the parameters described herein (e.g., percentage of wound epithelialization, epithelial thickness, granulation tissue formation, or area measurement (wound area)). A lack of substantial effect in an embodiment may include the absence of a statistically significant improvement in wound healing as determined by one or more of the parameters described herein.

[0083] The listing of ranges of values ​​in this document is intended only as a shorthand for individually referring to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were listed separately herein. The description of a range should be considered to have specifically disclosed all possible subranges and the individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 2.5, 2.7, 3, 4, 5, 5.1, 5.3, 5.8, and 6. Furthermore, any range referenced herein includes end values.

[0084] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of the group may be cited and claimed individually or in any combination with other members of the group or other elements found herein.

[0085] The following implementations and examples illustrate the exemplary methods provided herein. These examples are not intended to, and should not be construed as, limiting the scope of this disclosure. It will be apparent that these methods can be practiced in ways different from those specifically described herein. Many modifications and variations are possible in light of the teachings herein, and are therefore within the scope of this disclosure.

[0086] Exemplary embodiments The following are exemplary embodiments.

[0087] 1. A composition comprising an activated amphibian bud base, wherein the activated amphibian bud base has enhanced properties compared to a control group, and wherein the bud base is obtained from an injured amphibian less than 7 days after injury, and optionally, wherein the composition comprises a carrier.

[0088] 2. The composition according to embodiment 1, wherein the control group comprises unactivated bud bases obtained from amphibians, or wherein the control group comprises bud bases obtained from amphibians 7 days or more after injury.

[0089] 3. The composition according to embodiment 1 or 2, wherein the composition comprises a homogenized mixture of amphibian bud bases.

[0090] 4. The composition according to any one of embodiments 1 to 3, wherein the amphibian is a frog, toad, salamander or salamander.

[0091] 5. The composition according to any one of embodiments 1 to 4, wherein the amphibian is a larval amphibian, and optionally, wherein the larval amphibian includes a young frog, tadpole, caudal animals or larval anthropoid larvae.

[0092] 6. The composition according to any one of embodiments 1 to 5, wherein the amphibian is a larval tailed animal.

[0093] 7. The composition according to any one of embodiments 1 to 6, wherein the activated amphibian bud is obtained from the injury site between 12 hours and 7 days after the injury, 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days or 3 days after the injury.

[0094] 8. The composition according to any one of embodiments 1 to 7, wherein the composition is a pharmaceutical composition or a cosmetic composition, and the pharmaceutical composition or the cosmetic composition further comprises a pharmaceutically acceptable carrier or a cosmetically acceptable carrier.

[0095] 9. The composition according to any one of embodiments 1 to 8, wherein the composition further comprises one or more reagents that are heterologous to the activated bud base.

[0096] 10. The composition according to any one of embodiments 1 to 9, wherein the composition further comprises one or more agents comprising: peptides, proteins, pharmaceuticals, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or combinations thereof; and optionally, wherein one or more of the proteins or peptides comprise growth factors, cytokines, or chemokines; optionally, wherein one or more of the polymers comprise synthetic polymers or natural polymers or copolymers; optionally, wherein one or more of the pharmaceuticals comprise retinoic acid, corticosteroids, antifungal agents, antiviral agents, antibiotics, preservatives, local anesthetics, and antitumor agents; and optionally, wherein one or more of the pharmaceuticals comprise neomycin, polymyxin B, bacitracin, or combinations thereof.

[0097] 11. The composition according to any one of embodiments 1 to 10, wherein the composition is in the form of a gel, paste, solution, serum, cream, lotion, dispersion or emulsion.

[0098] 12. A method for preparing a composition comprising activated amphibian bud base, wherein the method comprises: Damage to amphibians; To enable the amphibian to recover from the injury; The bud base was separated from the injury site of the amphibian less than 7 days after the injury; and The composition comprising the activated amphibian bud base is prepared by activating the isolated bud base to induce the release of intracellular components from the bud base.

[0099] 13. The method according to embodiment 12, wherein activating the isolated bud base comprises: mechanical, chemical and / or electrical destruction that causes cell lysis to induce the release of intracellular components from the bud base.

[0100] 14. The method according to embodiment 12 or 13, wherein activating the separated bud base includes mechanical destruction, the mechanical destruction including: using a stirrer, homogenizer, sonicator or freeze-thaw process; or manually grinding the separated bud base, and optionally wherein the homogenizer is an ultrasonic homogenizer.

[0101] 15. The method according to any one of embodiments 12 to 14, wherein the activation of the isolated bud base includes chemical destruction, the chemical destruction including lysing the cells using anionic detergents, nonionic detergents or zwitterionic detergents.

[0102] 16. The method according to any one of embodiments 12 to 15, wherein activating the isolated bud base comprises using one or more detergents, the one or more detergents comprising sodium dodecyl sulfate (SDS, anionic), Triton X-100 (nonionic), Triton X-114 (nonionic), NP-40 (nonionic), Tween 20 (nonionic), Tween 80 (nonionic), CHAPS (zwitterionic), and CHAPSO (zwitterionic).

[0103] 17. The method according to any one of embodiments 12 to 16, wherein the activation of the isolated bud base includes electrical destruction, the electrical destruction including irreversible electroporation. 18. The method according to any one of embodiments 12 to 17, wherein the activation of the isolated bud base includes cell lysis or permeation lysis. 19. The method according to any one of embodiments 12 to 18, wherein activating the isolated bud base comprises lysing the cells in an aqueous saline solution, and optionally, wherein the saline solution is a phosphate buffer solution or a physiological saline solution.

[0104] 20. The method according to any one of embodiments 12 to 19, wherein the amphibian is a frog, toad, salamander, or fire salamander.

[0105] 21. The method according to any one of embodiments 12 to 20, wherein the amphibian is a juvenile amphibian, and optionally, wherein the juvenile amphibian includes young frogs, tadpoles, caudal animals or larval anthropoids.

[0106] 22. The method according to any one of embodiments 12 to 21, wherein the amphibian is a larval caudal avian.

[0107] 23. The method according to any one of embodiments 12 to 22, wherein the method includes separating the bud base from the injury site between 12 hours and 7 days after injury, 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days or 3 days after injury.

[0108] 24. The method according to any one of embodiments 12 to 23, wherein the injury to the amphibian includes any method for inducing bud formation.

[0109] 25. The method according to any one of embodiments 12 to 24, wherein the injury to the amphibian includes: amputation or tail amputation or creating a wound, and optionally, wherein the wound is a skin wound.

[0110] 26. The method according to any one of embodiments 12 to 25, wherein the method further comprises: preparing the activated bud base into a gel, paste, solution, serum, extract, cream, lotion, dispersion, powder or emulsion.

[0111] 27. A method for treating or preventing a skin disease or condition in a subject in need, the method comprising: administering to the subject a composition of an activated amphibian bud base as described in any one of embodiments 1 to 11, or the activated amphibian bud base obtained by the method described in any one of embodiments 12 to 26.

[0112] 28. The method according to implementation plan 27, wherein the skin condition includes fine lines and / or wrinkles, aging, redness, abrasions, burns, blisters, cuts, infections, razor bumps, scars, uneven skin tone, pain, stretch marks, improving skin elasticity and / or firmness, improving skin hydration, inflammation, or hyperpigmentation.

[0113] 29. The method described according to implementation plan 27 or 28, wherein preventing skin conditions includes: protecting the skin from damage caused by ultraviolet radiation and / or environmental pollution.

[0114] 30. The method according to any one of embodiments 27 to 29, wherein the method further comprises: treating the skin with a laser prior to applying the activated bud base, and optionally wherein the laser is an ablative fractional laser.

[0115] 31. The method according to any one of embodiments 27 to 30, wherein the skin condition is a burn, and the burn includes first-degree burns, second-degree burns or third-degree burns.

[0116] 32. The method according to any one of embodiments 27 to 30, wherein the skin condition is a wound, and the wound includes a deep dermal wound or a full-thickness wound.

[0117] 33. The method according to any one of embodiments 27 to 32, wherein the skin condition is a wound, and wherein the treatment with the composition enhances the epithelial thickness of the skin at the wound site, increases the re-epithelialization rate, increases the wound healing process, increases the wound closure efficiency, reduces leukocyte infiltration, and / or reduces scar formation.

[0118] Example Regeneration, the remarkable ability of organisms to replace missing or damaged tissues, has long captivated the imaginations of scientists and the public. Among nature's regenerative creatures, the axolotl (Mexican axolotl) stands out for its unparalleled regenerative capacity, serving as an exceptional model organism. From regenerating entire limbs to reconstructing complex organs, the axolotl has fascinated researchers for centuries.

[0119] Wound healing is a complex, multi-stage process involving synergistic interactions among various cell types, extracellular matrix components, and signaling molecules. Despite significant advances in medical science, chronic and non-healing wounds remain a major clinical challenge, affecting millions worldwide and placing a heavy burden on healthcare systems. Current treatments often fail to achieve complete functional tissue regeneration, highlighting the need for innovative therapeutic strategies.

[0120] This study demonstrates the results of using axolotl-derived gel to treat third-degree burns, deep dermal wounds, and full-thickness wounds in a porcine wound healing model. This research integrates insights from comparative biology, developmental genetics, and tissue engineering to explore the therapeutic potential of axolotls for human tissue regeneration. Furthermore, the researchers involved in this study chose to use in vivo models, particularly porcine wound healing models, for several reasons. Anatomically and physiologically, porcine skin is very similar to human skin, making it an ideal model for experimental research. The epidermal thickness of both porcine and human skin ranges from 50 to 120 micrometers. Considering the differences in epidermal thickness across different anatomical regions, the dermal-to-epidermal thickness ratio provides a more precise basis for comparison. In both species, this ratio consistently ranges from approximately 10:1 to 13:1, emphasizing the close similarity. In addition, both porcine and human skin exhibit well-defined reticular ridges and dermal papillae, as well as abundant subcutaneous adipose tissue. The biochemical similarity between porcine and human dermal collagen further supports the use of porcine skin in developing wound healing models, highlighting the model's relevance for translational research and therapeutic applications. Humans and pigs heal through similar physiological processes. Most small animals have a fascia and rely on wound contraction to close the wound. In contrast, humans and pigs close wounds through reepithelialization. Furthermore, the overall physiology of pigs is similar to that of humans, with most key organ systems being anatomically and functionally similar.

[0121] Example 1: Preparation of activated bud base This study used axolotls of different phenotypes. Animals were placed in dechlorinated water at 20°C to 22°C. For surgical procedures, axolotls were anesthetized with 0.1% ethyl 3-aminobenzoate methanesulfonate (Sigma, MS222), and the pH was adjusted to 7.0. Hind limbs were amputated below the knee or forelimbs below the elbow using a sterile scalpel (size 10).

[0122] All procedures were performed under the guidance of a licensed veterinarian to ensure that the animals were adequately sedated to minimize discomfort and to comply with ethical standards for the use of animals in research.

[0123] On day 0, the axolotl was injured by pruning its right forelimb and left hindlimb. Sprouts were harvested from the injured sites on days 1, 2, 3, 4, and 7, and these sprouts were subsequently activated for wound treatment. The harvested sprouts were sonicated according to the protocol outlined in Table 1. During sonication, the samples were kept cool in an ice bath to minimize thermal degradation. After sonication, the resulting gel was transferred to new vials and refrigerated until use.

[0124] Table 1: Ultrasound Procedure

[0125] Preparation for drug administration was made immediately before treating the pig wound model (described below) on the initial day of the experiment (Day 0). Subsequent doses were derived from the original formulation. Notably, the treatment materials were stored at 2°C to 8°C. This ensured that the materials used in all treatments were consistent with the initial formulation, maintaining the integrity of the experimental conditions.

[0126] Z gel is an activated bud harvested one day after injury to an axolotl. T gel is an activated bud harvested two days after injury to an axolotl. Y gel is an activated bud harvested three days after injury to an axolotl. F gel is an activated bud harvested four days after injury to an axolotl. X gel is an activated bud harvested seven days after injury to an axolotl.

[0127] The phrase "activated bud base on day #" refers to activated bud base prepared from bud base harvested on day # after injury. # is a number, such as 1, 2, 3, 4, or 7.

[0128] Example 2, Animal Research The aim of these studies was to determine the ability of activated bud bases to enhance healing using three different pig wound models.

[0129] Laboratory Animals. Pig models were used for experimental studies due to the morphological similarity between pig and human skin. Two (2) specific pathogen-free female pigs weighing 40-45 kg (from BG Looper Farm, 4673 Petra Mill Road, Granite Falls, NC28630) were housed indoors for at least 5 days prior to the start of the experiment to acclimatize. One animal suffered third-degree burns, and the other suffered deep dermal wounds as described below. These animals were fed a basal diet at random and housed individually in our animal facility (accredited by the American Association for Accreditation of Laboratory Animal Care [AAALAC]) with controlled temperature (19°C to 21°C) and light (12h / 12h LD).

[0130] wound model The wound models included a third-degree burn model, a deep dermal wound model, and a 20mm full-thickness wound model. Experiments A and B were conducted using these models.

[0131] Third-degree burn model. Figure 1An exemplary study design for third-degree burn wounds is shown. Twenty-five (25) third-degree burn wounds were created in the paravertebral and thoracic regions. A cauterizing iron (L&H Manufacturing Company, Mandan, North Dakota 58554) was used to create the burn wounds, with the heat controller set to 300°C. The iron was held vertically on the skin for 15 seconds, applying pressure by gravity to create burn wounds approximately 27 mm in diameter and 3 mm deep (to the subcutaneous tissue). The wounds were separated by 5 to 7 cm of unexfoliated skin. The wounds were randomly assigned to six treatment groups, with 3 or 4 wounds per treatment, such as... Figure 1 As shown. One of the three wounds was treated with laser before further treatment. The animal was treated within 20 minutes of wound formation.

[0132] Deep dermal wound model. Figure 2 An exemplary study design for deep dermal wounds is shown. Depth mesh-like dermal wound measurements (22mm × 22mm × 3mm, L × W × D) were made in the paravertebral and thoracic regions using a dedicated electric corneal knife equipped with a 22mm blade. Wounds were separated by 5cm to 7cm of uninjured skin. Wounds were randomly assigned to six treatment groups, with 3 or 4 wounds per treatment session. Figure 2 As shown. One of the three wounds was treated with laser before further treatment. The animal was treated within 20 minutes of wound formation.

[0133] A wound model with a full thickness of 20 mm. Figure 3 An exemplary study design for full-thickness wounds is shown. Full-thickness wounds were made in the paravertebral and thoracic regions using a 20mm circular biopsy puncturist. Figure 3 The wounds were separated by 5-7 cm of uninjured skin. The wounds were randomly assigned to seven treatment groups, with three wounds per treatment session. Figure 3 As shown. The animal was treated within 20 minutes of the wound formation.

[0134] Treatment plan Within the first hour following trauma, some wounds were treated with an erbium-YAG fractional ablation laser. This laser creates numerous micropillars of tissue ablation, enhancing the delivery and penetration of local medication into the wound. These pillars act as channels to enhance the delivery of local medication, allowing for better penetration into the injured area. For the micropulse energy, the laser was set at 15 mJ, a rate of 300 Hz, and a repetition delay of 30 seconds.

[0135] Following laser treatment, all third-degree burn wounds and deep dermal wounds were immediately treated with 200 μL of an activated bud-based gel formulation (prepared from buds harvested on days 1, 2, 3, 4, and 7). The gel formulation was applied using a sterile spatula to cover the injured area and surrounding unwound skin, and then covered with a Tegaderm dressing. All 20 mm full-thickness wounds received 100 μL of an activated bud-based gel formulation (prepared from buds harvested on days 1, 3, and 7 post-injury). Untreated control wounds in the animals were covered with a polyurethane film dressing (Tegaderm; 3M, St. Paul, MN). After application, all treatment groups were covered with Tegaderm.

[0136] All wounds were treated for 7 days (from day 0 to day 6 post-traumatic injury). After the treatment application, all wounds were covered with a polyurethane film dressing (Tegaderm; 3M, St. Paul, Minnesota). After the initial 7 days, the wounds were covered with non-adhesive gauze until final evaluation. On day 6 (7 days post-traumatic injury), all wounds received the final treatment application and were subsequently bandaged with non-adhesive gauze and Tegaderm. All dressings were secured with tape and covered with Coban self-adhesive elastic bandages (3M, St. Paul, MN).

[0137] Evaluate Histological evaluation. On the evaluation day (day 14 for third-degree burns, day 10 for deep dermal and full-thickness wounds), incisional biopsies were performed on each treatment group using a sterile scalpel. Biopsies were obtained through the center of the wound, including the adjacent normal skin on both sides. These specimens were placed in formalin and then stained with hematoxylin and eosin (H&E). One section of each tissue block was analyzed. The specimens were then scanned on an Olympus VS120 slide scanner, and the following parameters were evaluated in a blinded manner to determine the potential treatment response: Percentage of wound epithelialization (%) Measure the length of the wound surface that is covered by epithelium.

[0138] Epithelial thickness (cell layer μm) Epithelial thickness in biopsies may vary. The epithelial thickness (in micrometers (μm)) is measured at five equidistant points in the biopsy and the average value is taken.

[0139] Leukocyte infiltration The presence and number of mixed leukocyte infiltration in the subepithelial region were measured. The average scores were assigned as follows: 1 = none, 2 = mild, 3 = moderate, 4 = significant, 5 = severe.

[0140] Granulation tissue formationThe approximate gradation of the amount of new granulation tissue formed in the dermis is as follows: 0=0%, 0.5=1% to 10%, 1=11% to 30%, 2=31% to 50%, 3=51% to 70%, 4=71% to 90%, and 5=91% to 100%.

[0141] This comprehensive analysis provides important insights into its efficacy in promoting wound healing, epithelial regeneration, and inflammatory responses.

[0142] Histological results.

[0143] Data were pooled and analyzed to determine treatment response. The percentage of epithelialization and other histological parameters in the wound were plotted against the number of days post-treatment. Assessments for third-degree burns, deep dermal burns, and full-thickness burns were performed on days 14, 10, and 10, respectively. Since there were only one, two, or three samples per treatment group, only average values ​​were used in tables and graphs for illustrative purposes.

[0144] Percentage of epithelialization. The percentage of epithelialization represents the area of ​​the wound region covered by newly formed epidermis with one or more layers of keratinocytes. This is a good indicator of the rate of keratinocyte migration and the first step of epithelialization. Figure 4 to Figure 14 The percentage of epithelialization in different trials using three different wound models treated with activated bud substrate harvested from bud substrate harvested on days 1, 2, 3, 4, and 7 post-injury is shown compared to the control group. The results indicate that treatment with activated bud substrate harvested less than 7 days post-injury in *Axolotl*, and subsequently activated, can be used with or without laser pretreatment to effectively treat various wounds.

[0145] Epithelial thickness. Epithelial thickness is a measure of the average thickness of five points on newly formed epithelium. Epithelial thickness reflects the processes of keratinocyte proliferation, differentiation, and epidermal maturation. Figure 21 and Figure 22 ).

[0146] White cell infiltration (WCI). WCI is used to assess inflammatory responses. Figure 15 , Figure 16 , Figure 23 and Figure 24 ).

[0147] Granulation tissue formation. Dermal reconstruction begins approximately 3 to 4 days after injury, marked by granulation tissue formation. Dermal reconstruction includes neovascularization (angiogenesis), fibroblast accumulation, and collagen extracellular matrix accumulation. Granulation tissue formation is measured as the percentage of the wound surface filled with newly formed granulation tissue. Figure 17 , Figure 18, Figure 25 and Figure 26 ).

[0148] Area measurement method Wound areas from each treatment group at each time point (every 3 to 4 days) were pooled and analyzed for quantitative analysis. Wound areas from different treatments using activated budding materials (prepared from budding materials harvested on days 1, 2, 3, 4, and 7 post-injury) were compared after laser application to third-degree burn wounds. At day 14, the wound area was lower than that of the untreated control group, confirming the effectiveness of activated budding materials in wound treatment (Figures 27 to 28). Figure 30 ).

[0149] Clinical observation (erythema).

[0150] Erythema – indicates the degree of inflammation present. *Score: 1 = None, 2 = Mild, 3 = Moderate, 4 = Significant, 5 = Severe Throughout the study, none of the wounds on any of the animals showed erythema (no scoring).

[0151] Digital photography and wound contraction measurement. Each group of wounds was photographed, and the wound area was tracked to measure wound contraction. Digital imaging was performed using ImageJ (a software tool developed by the National Institutes of Health (NIH)) to carefully delineate the wound circumference. ImageJ is widely used in the scientific community for image analysis due to its powerful features and user-friendly interface.

[0152] To assess wound contraction, the circumference of each wound was tracked and compared with measurements taken on day 0. This comparison allows for precise quantification of the extent of wound contraction over time. ImageJ's ability to handle large datasets and its advanced image processing algorithms make it ideal for this analysis, ensuring the accuracy and reproducibility of our measurements.

[0153] result Third-degree burns. Without supplemental laser treatment, third-degree burns treated with activated bud bases on day 3 achieved 100% re-epithelialization by day 14. Notably, this was the only treatment to achieve complete re-epithelialization during the study period. In contrast, the untreated control group without laser exposure achieved 76.20% re-epithelialization at the same time point. In the non-laser treatment groups, the activated bud base group on day 1 (47.95%) resulted in the lowest re-epithelialization rate at day 14, followed by the activated bud base group on day 7 (48.3%). Figure 9A1 Nevertheless, when laser treatment was included as part of the overall treatment regimen, reepithelialization increased by more than 50% in the latter two groups. Figure 9A2The opposite was true for the bud base groups activated on day 2 and day 3; in both groups, supplemental laser therapy had an antagonistic effect.

[0154] The results showed that on day 14 without laser treatment, the epithelial thickness in third-degree burns varied depending on the time of the test specimens (activated bud bases) (i.e., day 1, day 2, day 3, day 4, or day 7 after bud base induction). The activated bud base group on day 2 had the highest epithelial thickness at 122.1 μm, followed by the activated bud base group on day 7 with a thickness of 118.1 μm. Figure 13 The activated bud base group on day 1 showed a thickness of 99.75 μm, while the activated bud base groups on day 3 and day 4 showed thicknesses of 81.35 μm and 82.5 μm, respectively. The untreated control group showed an epithelial thickness of 87.95 μm.

[0155] In the group treated with laser therapy prior to activated bud base therapy, epithelial thickness resulted in a denser distribution. The activated bud base group showed the thickest epithelium at day 14, measuring 112.8 µm ( ). Figure 14 Following closely were the untreated control group and the activated bud base group on day 2, with measurements of 110.7 µm and 107.9 µm, respectively. The activated bud base group on day 1 produced the thinnest epithelial measurement, at 76.5 µm.

[0156] The activated bud base group on day 7 showed the highest score for leukocyte infiltration, at 3.5, followed by the activated bud base group on day 1, which scored 3 ( ) on non-laser wounds. Figure 15 On day 3, the activated bud base group and the untreated control group (without laser treatment) had the lowest scores, both at 2. On the other hand, activated bud base treatment supplemented with laser therapy showed a decrease in leukocyte infiltration scores, with scores of 3 in both groups except for the activated bud base groups on day 2 and day 3. Figure 16 ).

[0157] When comparing treatments with and without laser supplementation, granulation tissue scores showed a similar trend to leukocyte infiltration scores. Specifically, in the absence of laser treatment, the granulation tissue scores were lowest in the day 2 and day 3 activated granulation tissue groups, both at 2.5, followed by the untreated control group with a score of 3. Figure 17 In contrast, without laser supplementation, the granulation tissue score of the activated bud base group on day 4 and day 7 consistently reached 3.5. Figure 17It is noteworthy that when laser therapy was introduced, the granulation tissue treatment on day 2 and day 3 after activation of the bud base significantly increased to 4, a significant increase of 1.5 points. Figure 18 However, when laser treatment was included, the granulation tissue after activating the bud base treatment on day 7 decreased to 2 ( Figure 18 ).

[0158] Deep dermal wounds. Similar to findings in the grade III wound model, in the absence of laser, activated budding tissue on day 3 led to 100% reepithelialization in deep dermal wounds by day 10. Figure 19 The results showed that, on day 2, the activated bud base group resulted in 86.1% re-epithelialization on day 10; on day 4, the activated bud base group resulted in 85.4% re-epithelialization on day 10; the untreated control group resulted in 78% re-epithelialization on day 10; the activated bud base group on day 7 resulted in 70.6% re-epithelialization on day 10; and the activated bud base group on day 1 resulted in 60.9% re-epithelialization on day 10. Figure 19 Prescribing laser treatment before activated bud base therapy produced different results. For wounds receiving laser treatment, the results showed that re-epithelialization was 100% in the activated bud base group on day 1, 95.8% in the untreated control group, 90.8% in the activated bud base group on day 3, 85.5% in the activated bud base group on day 4, 63.1% in the activated bud base group on day 7, and 62.4% in the activated bud base group on day 2. Figure 20 ).

[0159] On day 10 post-injury, without laser treatment, the epithelial thickness of the deep dermal wound varied among different activated budding sites. The activated budding site group showed the highest epithelial thickness on day 4, measured at 127.7 μm. Figure 21 The second group was the activated bud base group on day 1, which showed an epithelial thickness of 117 μm. Figure 21 The activated bud base groups on day 7 and day 3 showed thicknesses of 105.65 μm and 100.35 μm, respectively. Figure 21 In the absence of laser treatment, the epithelial thickness of the untreated control group was 97.4 µm. Figure 16 The lowest epithelial thickness was observed in the activated bud sac group on day 2, measured at 78.2 µm. Figure 21 ).

[0160] On the other hand, in the case of laser treatment, the epithelial thickness of the deep dermal wound on day 10 post-injury showed that the activated budding tissue group on day 7 had the highest epithelial thickness, measured at 157.4 μm, followed by the activated budding tissue group on day 2 with an epithelial thickness of 140.3 μm. Figure 22 In the case of laser treatment, the thickness in the control group was 93.4 µm. Figure 22 The epithelial thickness of the activated bud base group on day 3 and the activated bud base group on day 1 was 90.6 μm and 77.7 μm, respectively. Figure 22 The lowest epithelial thickness was observed in the activated bud base group on day 4, measured at 65.7 µm.

[0161] On day 10 post-injury, without laser treatment, leukocyte infiltration (WCI) in the deep dermal wound showed that the activated bud base group on day 1 exhibited the highest WCI score of 4 (23), indicating a significant inflammatory response. In contrast, all other activated bud base treatments (activated bud base group on day 2, day 3, day 4, and day 7) had a consistent WCI score of 2 ( Figure 23 Without laser treatment, the control group showed the lowest WCI score, at 1.5 ( ). Figure 23 For laser treatment, all activated bud base treatment groups scored 1, while the untreated laser-treated control group scored 3, which was twice the score recorded in the untreated, non-laser-treated control group. Figure 24 ).

[0162] On the 10th day post-injury, without laser treatment, granulation tissue formation in the deep dermal wound showed that both the activated bud base group on day 1 and the activated bud base group on day 2 exhibited the highest granulation tissue score, which was 2 ( Figure 25 On day 4 and day 7, the granulation tissue score of the activated bud base group was 1.5, while the lowest granulation tissue score, 1, was observed in the activated bud base group on day 3 and the untreated control group. Figure 25 In deep dermal wounds treated with laser prior to activated budding therapy, consistency was observed in most activated budding therapy groups. Activated budding therapy on day 1, day 2, day 4, and day 7 all resulted in the highest granulation tissue score, 2 (…). Figure 26 In contrast, the untreated laser-treated group and the activated bud base group treated with laser on day 3 showed lower granulation tissue scores, with a score of 1 ( ). Figure 26 ).

[0163] discuss The results of this study elucidate the significant potential of caudal bud-based products (such as axolotl bud-based products) in promoting wound healing, particularly in challenging conditions such as third-degree burns and deep dermal wounds in porcine models. This research provides important insights into the benefits and mechanisms of using this regenerative therapy, with a particular focus on re-epithelialization, epithelial thickness, leukocyte infiltration, and granulation tissue formation.

[0164] The results showed a significant advantage in re-epithelialization when using activated budding material. Notably, third-degree burn wounds treated with activated budding material exhibited a re-epithelialization rate as high as 100% on day 14 post-injury (third-degree burn), significantly higher than the untreated control group. Similar results were observed in deep dermal wounds, although less pronounced. This suggests that the combination of activated budding material and laser therapy can significantly accelerate the wound healing process for third-degree burns, as well as potentially deep dermal and full-thickness wounds. Furthermore, the results obtained from activated budding material harvested at different time points after budding induction (post-injury) provide evidence of the importance and complexity of timely harvesting and application to subjects in regenerative therapy.

[0165] Regarding re-epithelialization, the activated bud base treatment group, especially the activated bud base group on day 3, demonstrated significant wound closure efficiency. By day 14, re-epithelialization of third-degree burns reached 100%, highlighting the significantly enhanced skin regeneration capacity of the activated bud base. In contrast, untreated wounds showed poor results, demonstrating the unique regenerative potential of the activated bud base.

[0166] Variables in epithelial thickness were observed along the treatment timeline, with the thickest new epithelium observed in the activated bud base group on day 2. The introduction of laser therapy altered epithelial outcomes, typically increasing thickness, particularly in samples from day 7. These variations underscore the importance of timing and sequence in regeneration protocols.

[0167] Epithelial thickness is a key indicator of the quality and robustness of healing skin. Results showed that non-laser treatment generally resulted in a thicker epithelial layer compared to laser treatment. For example, in third-degree burns, the activated bud base group on day 2 without laser treatment achieved an epithelial thickness of 122.1 µm, which was superior to the 76.5 µm observed in the activated bud base group on day 1 with laser treatment. A similar trend was observed in deep dermal wounds.

[0168] Reduced leukocyte infiltration was observed in some combinations, leading to improved overall wound healing. Certain laser-treated groups showed reduced inflammation, as evidenced by lower leukocyte infiltration scores, suggesting a potential anti-inflammatory effect in the combination of laser and mexillary axilla treatment. This aspect is crucial for minimizing scarring and improving overall healing quality.

[0169] Non-laser-treated wounds appear to allow treatment to be more effectively focused on the superficial layers of skin, rather than penetrating deeper tissues that are facilitated by the microchannels created during the laser process.

[0170] Granulation tissue formation is crucial for successful wound healing as it provides a scaffold for new tissue growth. Granulation tissue formation was relatively consistent across the treatment groups, but varied slightly depending on the treatment method. Interestingly, non-laser-treated wounds generally exhibited better granulation tissue formation, which may be attributed to the less invasive nature of the treatment, allowing for a more natural healing process. Finally, the study showed that wounds treated with activated budding tissue, particularly those combined with laser therapy, exhibited reduced leukocyte infiltration. For example, on day 1 after laser treatment, the leukocyte infiltration score in the activated budding tissue group was 2, while the score in the untreated, non-laser group was 3. This reduction in inflammation suggests that activated budding tissue can help modulate the immune response, potentially leading to less scarring and faster recovery.

[0171] In summary, activated bud-based products derived from axolotls, particularly when combined with laser therapy, show great promise in promoting the healing of third-degree burns, deep dermal wounds, and full-thickness wounds. The significant improvements in re-epithelialization and inflammation reduction suggest that this approach could be a valuable addition to current wound care practices, offering new hope to patients with severe and problematic wounds.

[0172] The foregoing subject matter is provided by way of illustration only and should not be construed as limiting. Various modifications and changes may be made to the subject matter described herein without following the exemplary embodiments and applications illustrated and described, and without departing from the true spirit and scope of this disclosure as set forth in the appended claims.

[0173] All publications, patents, and patent applications referenced in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Although various embodiments have been described above, those skilled in the art will understand that various modifications, substitutions, omissions, and alterations can be made without departing from their spirit.

[0174] References Demircan et al. J. Exp. Clin. Med. 2016, 33(4): 229-237 WO 2020 / 032902

Claims

1. A composition comprising activated amphibian bud base, wherein, The activated amphibian bud base includes enhanced properties compared to the control group, and wherein the bud base is obtained from an injured amphibian less than 7 days after injury, and optionally, wherein the composition includes a carrier.

2. The composition according to claim 1, wherein, The control group includes unactivated buds obtained from amphibians, or wherein the control group includes buds obtained from amphibians 7 days or more after injury.

3. The composition according to claim 1, wherein, The composition comprises a homogenized mixture of amphibian bud bases.

4. The composition according to claim 1, wherein, The amphibians mentioned are frogs, toads, salamanders, or salamanders.

5. The composition according to claim 1, wherein, The amphibians are juvenile amphibians, and optionally, the juvenile amphibians include young frogs, tadpoles, caudal animals, or larval anthropoid larvae.

6. The composition according to claim 1, wherein, The amphibians described are juvenile tailless animals.

7. The composition according to claim 1, wherein, The activated amphibian buds were obtained from the injury site between 12 hours and 7 days after the injury, 1 to 6 days after the injury, 1 to 5 days after the injury, 1 to 4 days after the injury, 2 to 4 days after the injury, or 3 days after the injury.

8. The composition according to claim 1, wherein, The composition is a pharmaceutical composition or a cosmetic composition, and the pharmaceutical composition or the cosmetic composition further includes a pharmaceutically acceptable carrier or a cosmetically acceptable carrier.

9. The composition according to claim 1, wherein, The composition further includes one or more reagents that are heterologous to the activated bud base.

10. The composition according to claim 1, wherein, The composition further comprises one or more agents comprising: peptides, proteins, pharmaceuticals, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or combinations thereof; and optionally, wherein one or more of the proteins or peptides comprise growth factors, cytokines, or chemokines; optionally, wherein one or more of the polymers comprise synthetic polymers or natural polymers or copolymers; optionally, wherein one or more of the pharmaceuticals comprise retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, preservatives, local anesthetics, and antitumor drugs; and optionally, wherein one or more of the pharmaceuticals comprise neomycin, polymyxin B, bacitracin, or combinations thereof.

11. The composition according to claim 1, wherein, The composition is in the form of a gel, paste, solution, serum, cream, lotion, dispersion, or emulsion.

12. A method for preparing a composition comprising activated amphibian bud base, wherein, The method includes: Damage to amphibians; To enable the amphibian to recover from the injury; The bud base was separated from the injury site of the amphibian less than 7 days after the injury; and The composition comprising the activated amphibian bud base is prepared by activating the isolated bud base to induce the release of intracellular components from the bud base.

13. The method according to claim 12, wherein, The activation of the isolated bud base includes: mechanical, chemical and / or electrical disruption that causes cell lysis to induce the release of intracellular components from the bud base.

14. The method according to claim 12, wherein, The activation of the isolated bud base includes mechanical disruption, which may include: using a stirrer, homogenizer, sonicator, or freeze-thaw process; or manually grinding the isolated bud base, and optionally wherein the homogenizer is an ultrasonic homogenizer.

15. The method according to claim 12, wherein, The activation of the isolated germinal base includes chemical destruction, which includes lysing the cells using anionic, nonionic, or zwitterionic detergents.

16. The method according to claim 12, wherein, The activation of the isolated bud base includes the use of one or more detergents, including sodium dodecyl sulfate (SDS, anionic), Triton X-100 (nonionic), Triton X-114 (nonionic), NP-40 (nonionic), Tween20 (nonionic), Tween80 (nonionic), CHAPS (zwitterionic), and CHAPSO (zwitterionic).

17. The method according to claim 12, wherein, The activation of the isolated bud base includes electrical destruction, which includes irreversible electroporation.

18. The method according to claim 12, wherein, The activated and isolated bud base includes cell lysis or osmotic lysis.

19. The method according to claim 12, wherein, The activation of the isolated bud base includes lysing the cells in an aqueous saline solution, and optionally, the saline solution is a phosphate buffer solution or a physiological saline solution.

20. The method according to claim 12, wherein, The amphibians mentioned are frogs, toads, salamanders, or salamanders.

21. The method according to claim 12, wherein, The amphibians are juvenile amphibians, and optionally, the juvenile amphibians include young frogs, tadpoles, caudal animals, or larval anthropoid larvae.

22. The method according to claim 12, wherein, The amphibians described are juvenile tailless animals.

23. The method according to claim 12, wherein, The method includes separating bud bases from the injury site between 12 hours and 7 days after the injury, 1 to 6 days after the injury, 1 to 5 days after the injury, 1 to 4 days after the injury, 2 to 4 days after the injury, or 3 days after the injury.

24. The method according to claim 12, wherein, The damage to the amphibian includes any method that induces bud formation.

25. The method according to claim 12, wherein, The injury to the amphibian includes: amputation or tail amputation or causing a wound, and optionally, wherein the wound is a skin wound.

26. The method according to claim 12, wherein, The method further includes: preparing the activated bud base into gels, pastes, solutions, serums, extracts, creams, lotions, dispersions, powders, or emulsions.

27. A method for treating a wound of a subject, or for treating or preventing a skin disease or condition of a subject, said method comprising: The subject is given a composition of the activated amphibian bud base according to any one of claims 1 to 11 or the activated amphibian bud base obtained by any one of claims 12 to 26.

28. The method according to claim 27, wherein, The skin conditions mentioned include fine lines and / or wrinkles, aging, redness, abrasions, burns, blisters, cuts, infections, razor bumps, scars, uneven skin tone, pain, stretch marks, improving skin elasticity and / or firmness, improving skin hydration, inflammation, or hyperpigmentation.

29. The method according to claim 27, wherein, Preventing skin conditions includes protecting the skin from damage caused by ultraviolet radiation and / or environmental pollution.

30. The method according to claim 27, wherein, The method further includes treating the skin with a laser prior to applying the activated bud base, and optionally wherein the laser is an ablative fractional laser.

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