Activated amphibian cells and uses thereof

Abstract

The present disclosure describes compositions comprising activated amphibian blastemas that are useful in treating different skin diseases and conditions. Blastemas harvested from the injury site of amphibians 1 to 7 days after injury and subsequently activated were effective in treating skin diseases and conditions.

Description

ACTIVATED AMPHIBIAN CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 602,257, filed November 22, 2023, which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present disclosure describes compositions comprising activated amphibian blastema cells and uses in the treatment of wounds. BACKGROUND

[0003] The skin, the largest organ of the mammalian body, is an outer covering of the body that serves three main functions: protection against microbes and excessive water loss, regulation of body temperature, and sensation of touch, heat, and cold. Mammalian skin has two primary layers, the epidermis, and the dermis. The epidermis, the outermost layer of the skin that prevents microbes from entering and keeps water in our body, is a stratified squamous epithelium composed of keratinocytes. The dermis is the layer of skin beneath the epidermis which serves as a location for the appendages of the skin and provides elasticity to the skin through an extracellular matrix composed of collagen fibers, elastic fibers, hyaluronan, and proteoglycans. The dermis and the 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 the remodeling, repair, and regeneration process. Underneath the dermis is the hypodermis which is composed of loose connective tissue, such as fat, and elastin. The primary cells of the hypodermis include fibroblasts, macrophages, and adipocytes.

[0004] An injury or a disease creates an interruption of the morphology and function of an organ or tissue, such as the skin. The remodeling and repair of the organ or tissue following an injury is a complex wound healing process involving interactions between cells, growth factors, and extracellular matrix (ECM). The process in adult mammals involves well-known stages: homeostasis, inflammation, proliferation, maturation, and remodeling. During homeostasis, clotting takes place to stop the 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 tissues, such as new connective tissues and blood vessels, replace the wound. Maturation and remodeling take place after the wound is closed and involve the repair of the dermal tissues to improve their tensile strength.

[0005] In contrast to the repair process in which the goal is to re-establish function without regard to the exact placement of injured tissue, regeneration is the replacement of injured tissuewith an exact copy such that both morphology and functionality are completely restored. As an example, non-injured skin undergoes complete regeneration continually with the replacement of new cells. However, injured adult mammalian skin does not regenerate completely and heals with a scar.

[0006] Although all mammals including humans can spontaneously regenerate the tips of their fingers into adult life, mammals are not able to regenerate their limbs in contrast to amphibians. Most amphibians can regenerate missing body parts. However, their regenerative ability to regenerate varies extensively from species to species. Urodeles, for example, have exceptional regenerative capabilities.

[0007] In amphibians, in response to amputation, cells from the limb tissue around the amputation surface dedifferentiate and give rise to a mass of undifferentiated and proliferating cells called blastema cells, which are capable of regenerating into a body part. Some amphibians can produce blastemas as adults enabling them to regenerate even as adults. Blastemas are important in regeneration.

[0008] Adult mammalian skin wound repair commonly results in scar tissue formation. Unlike mammals, amphibians repair wounds by regeneration instead of scarring. It is desirable to develop methods for treating mammalian wounds that would not lead to scarring and in a shortened period. SUMMARY

[0009] This Summary is provided to introduce a selection of concepts in a simplified form that is further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0010] The present disclosure provides compositions comprising activated blastemas. The blastemas are obtained from an injured amphibian less than 7 days after injury. In embodiments, the amphibian is a neotenic Urodele, such as an axolotl.

[0011] The present disclosure also provides methods of preparing the activated blastemas comprising injuring an amphibian, allowing the amphibian to recover from the injury, isolating blastemas from the injury site of the amphibian less than 7 days after the injury, and activating the isolated blastemas to induce release of intracellular components from the blastema cells. Activating the isolated blastemas includes mechanical or chemical means including homogenization, using detergent, or cytolysis to lyse the blastemas.

[0012] The present disclosure further provides treating or preventing a skin disease or condition or a wound in a subject with the activated blastema. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1: Experimental Design for Third-Degree Burn Animal. Gel Z, T, Y, F, or X was applied to the animal after injury (third-degree burn injury) at each of the sites. H is the untreated control. Gel Z (PowerBlast 1 day) is activated blastema harvested one day after injury of the amphibian and was applied to wounds #3, #12, and #13. Gel T (PowerBlast 2 days) is activated blastema harvested two days after injury of the amphibian and was applied to wounds #4, #5, and #14. Gel Y (PowerBlast 3 days) is activated blastema harvested three days after injury of the amphibian and was applied to wounds #8, #9, and #22. Gel F (PowerBlast 4 days) is activated blastema harvested four days after injury of the amphibian and was applied to wounds #18, #21, and #23. Gel X (PowerBlast 7 days) is activated blastema harvested seven days after injury of the amphibian and was applied to wounds #1, #7, and #19. Wounds #10, #15, and #24 were untreated controls (H). All wounds were treated for 7 days (from initial wounding Day 0 until Day 6). All wounds were covered after treatment application with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) until Day 11, the Tegaderm dressing was replaced with non- adherent gauze until the final assessment day (Day 14 after wounding). In each treatment group, two wounds were treated with topical application and the third wound (bar) included laser-assisted delivery (LAD). Wounds #5, #9, #12, #15, #18, and #19 were lasered.

[0014] FIG.2: Experimental Design for Deep Dermal Wound Animal. Gel Z, T, Y, F, or X was applied to the animal after injury (deep dermal injury) at each of the sites. H is the untreated control. Gel Z is activated blastema harvested one day after injury of the amphibian and was applied to wounds #3, #14, and #12. Gel T is activated blastema harvested two days after injury of the amphibian and was applied to wounds #4, #15, and #25. Gel Y is activated blastema harvested three days after injury of the amphibian and was applied to wounds #8, #9, and #20. Gel F is activated blastema harvested four days after injury of the amphibian and was applied to wounds # 5, #19, and #22. Gel X is activated blastema harvested seven days after injury of the amphibian and was applied to wounds #1, #7, and #16. Wounds 10, 21, and 24 were untreated controls (H). All wounds were treated for 7 days (from initial wounding Day 0 until Day 6). All wounds were covered after treatment application with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) until the final assessment day (Day 10 after wounding). In each treatment group, two wounds were treated with topical application and the third wound (bar) included laser-assisted delivery (LAD). Wounds #1, #4, #9, #14, #22, and #24 were lasered.

[0015] FIG.3: Experimental Design for 20 millimeters (mm) Full thickness Wound Animal. Gel X, Y, or Z was applied to the animal after injury (deep dermal injury) at each of the sites. H is the untreated control. Gel X is activated blastema harvested seven days after injury of the amphibian and was applied to wounds #1, #7, and #23. Gel Y is activated blastema harvested three days after injury of the amphibian and was applied to wounds #8, #9, and #18. Gel Z is activated blastema harvested one day after injury of the amphibian and was applied to wounds #3, #13, and #24. Wounds 12, 19, and 22 were untreated controls (H). In each treatment group, twowounds were treated with topical application and the third wound (encircled) included laser- assisted delivery (LAD). Wounds #13, #18, #19, and #23 were lasered.

[0016] FIGs. 4A1-4C: Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds. Gel Z (one day) was used to treat the different wounds in trials B and A. (4A1 and 4A2) % epithelialization of third-degree burn wounds for trials B and A with and without laser pretreatments. (4B) % epithelialization of deep dermal wounds for trial B with and without laser pretreatment. (4C) % epithelialization of full thickness wounds for trial A with and without laser pretreatment.

[0017] FIGs. 5A-5B: Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds. Gel T (two days) was used to treat the different wounds in trial B. (5A) % epithelialization of third-degree burn wounds for trial B with and without laser pretreatments. (5B) % epithelialization of deep dermal wounds for trial B with and without laser pretreatment.

[0018] FIGs. 6A1-6C: Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds. Gel Y (three days) was used to treat the different wounds in trials B and A. (6A1 and 6A2) % epithelialization of third-degree burn wounds for trials B and A with and without laser pretreatments. (6B) % epithelialization of deep dermal wounds for trial B with and without laser pretreatment. (6C) % epithelialization of full thickness wounds for trial A with and without laser pretreatment.

[0019] FIGs. 7A1-7B2: Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds. Gel F (four days) was used to treat the different wounds in trial B. (7A1 and 7A2) % epithelialization of third-degree burn wounds for trial B with and without laser pretreatments. (7B1 and 7B2) % epithelialization of deep dermal wounds for trial B with and without laser pretreatment.

[0020] FIGs. 8A1-8C: Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds. Gel X (seven days) was used to treat the different wounds in trials B and A. (8A1 and 8A2) % epithelialization of third-degree burn wounds for trials B and A with and without laser pretreatments. (8B) % epithelialization of deep dermal wounds for trial B with and without laser pretreatment. (8C) % epithelialization of full thickness wounds for trial A with and without laser pretreatment.

[0021] FIGs.9A1-9B2: Average Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds of Trial B. (9A1 and 9A2) Average % epithelialization of third-degree burn wounds for trial B with and without laser pretreatments. (9B1 and 9B2) Average % epithelialization of deep dermal wounds for trial B with and without laser pretreatment.

[0022] FIGs.10A1-10B2: Average Percent (%) Epithelialization of Third-Degree Burn Wounds and Full Thickness Wounds of Trial A. (10A1 and 10A2) Average % epithelialization of third-degree burn wounds for trial A with and without laser pretreatments. (10B1 and 10B2) Average % epithelialization of full thickness wounds for trial A with and without laser pretreatment.

[0023] FIGs.11A-11D: Percent (%) Epithelialization of Third-Degree Burn Wounds and Deep Dermal Wounds of Trial B. Gels X, F, Y, T, and Z were used to treat third-degree burn wounds and deep dermal wounds in Trial B. (11A) % epithelialization of third-degree burn wounds for trial B without laser pretreatment. (11B) Average % epithelialization of third-degree burn wounds for trial B without laser pretreatments. (11C) % epithelialization of deep dermal wounds for trial B without laser pretreatment. (11D) Average % epithelialization of deep dermal wounds for trial B without laser pretreatment

[0024] FIGs 12A1-12B2: Average Percent (%) Epithelialization of Third-Degree Burn Wounds, Deep Dermal Wounds, and Full Thickness Wounds in Trials A and B. (12A1 and 12A2) Average % epithelialization of third-degree burn wounds for trials A and B with and without laser pretreatments. (12B1 and 12B2) Average % epithelialization of deep dermal wounds and full thickness wounds for trials A and B with and without laser pretreatments.

[0025] FIG. 13: Epithelial Thickness of Third-Degree Burn Wounds at Day 14 Post Injury Without Laser Treatment.

[0026] FIG.14: Epithelial Thickness of Third-Degree Burn Wounds at Day 14 Post Injury With Laser Treatment.

[0027] FIG. 15: White Cell Infiltration of Third-Degree Burn Wounds at Day 14 Post Injury Without Laser Treatment

[0028] FIG. 16: White Cell Infiltration on Third-Degree Burn Wounds at Day 14 Post Injury With Laser Treatment

[0029] FIG.17: Granulation tissue of Third-Degree Burn Wounds at Day 14 Post Injury Without Laser Treatment.

[0030] FIG.18: Granulation tissue of Third-Degree Burn Wounds at Day 14 Post Injury With Laser Treatment.

[0031] FIG.19: Percent Re-Epithelialization of Deep Dermal Wounds at Day 10 Post Injury Without Laser Treatment.

[0032] FIG.20: Percent Re-Epithelialization of Deep Dermal Wounds at Day 10 Post Injury With Laser Treatment.

[0033] FIG.21: Epithelial Thickness on Deep Dermal Wounds at Day 10 Post Injury Without Laser Treatment.

[0034] FIG.22: Epithelial Thickness on Deep Dermal Wounds at Day 10 Post Injury With Laser Treatment.

[0035] FIG.23: White Cell Infiltration on Deep Dermal Wounds at Day 10 Post Injury Without Laser Treatment.

[0036] FIG. 24: White Cell Infiltration on Deep Dermal Wounds at Day 10 Post Injury With Laser Treatment.

[0037] FIG.25: Granulation Tissue Formation of Deep Dermal Wounds at Day 10 Post Injury Without Laser Treatment.

[0038] Figure 26: Granulation Tissue Formation of Deep Dermal Wounds at Day 10 Post Injury With Laser Treatment.

[0039] FIGs.27A and 27B: Decreasing Wound Area of Treated Third-Degree Burn Wounds With Time. (27A) Wound area measurements from Day 0 to Day 14. (27B) Third-Degree Burn Wounds Measurements With Laser Treatment.

[0040] FIG.28: Third-Degree Burn Wounds Measurements Without Laser Treatment.

[0041] FIG.29: Deep Dermal Wounds Measurements With Laser Treatment.

[0042] FIG.30: Deep Dermal Wounds Measurements Without Laser Treatment. DETAILED DESCRIPTION

[0043] In contrast to mammals, amphibians, such as Urodeles, regenerate their skin structures including the dermis and secretion glands without forming a scar after a deep skin injury. Demircan reported that amphibian tissues can heal wounds in mice. Demircan only discloses using amphibian blastema tissue graft or blastema cells on wounds. Dermircan describes the blastema stage as between 24 hours to 4 weeks and preferably using the blastema on day 7, day 10, or day 15 after amputation (WO 2020034902).

[0044] Accordingly, it was surprising to discover that blastemas harvested from a site of injury of an amphibian less than 7 days after injury and subsequently activated have wound healing potential. In embodiments, activated blastemas prepared from amphibian blastemas 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 have wound healing potential. In embodiments, blastema cells can be activated to have enhanced wound healing potential 1 to 5 days, 2 to 4 days, or 3 days after injury.

[0045] The terms “a,” “an,” “the,” and similar referents used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0046] The term “activated blastemas” refers to blastemas harvested from amphibians a certain number of days after injury and subsequently activated to induce the release of intracellular components from the blastema cells.

[0047] The term “skin conditions” includes skin conditions that require therapeutic “drug” treatment including diseases, defects, and injuries including wounds, such as burns, deep dermal wounds, and full thickness wounds.

[0048] The term “cosmetic skin conditions” includes skin conditions that are related to tone, clarity, radiance, brightness, and / or hydration of the skin.

[0049] The term "subject" refers to an animal, for example, a mammal. Examples of mammals include a human, a dog, a cat, a horse, a cow, a goat, a sheep, a pig, or a non-human primate. A subject in need of treatment or a subject in need thereof includes a subject having a disease or condition that needs to be treated. A subject in need thereof includes a subject that needs treatment and / or prevention of a skin condition or a subject that has a wound that needs to be treated.

[0050] The term "therapeutically effective amount" refers to an amount of a product or composition that provides a therapeutic benefit in the treatment, prevention, or management of a condition or disease, such as a skin disease, an injury to the skin, or a wound, for example, a drug product. The term “therapeutically effective amount” also includes the amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated.

[0051] 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 desirable clinical / medical end-point, including alleviating symptoms of a disease or condition. Examples of such desirable end-points associated with skin disease or condition include wound healing, tissue closure, bulking tissue, preventing tissue adhesion, providing structural support to tissue, providing a protective barrier, and / or correcting a defect. Administering the product also includes applying the product to a subject.

[0052] The term “xenogenic” refers to a product derived or originated from a member of another species.

[0053] The term “amphibians” refers to cold-blooded vertebrate animals that include frogs, toads, newts, and salamanders. They have an aquatic gill-breathing larval stage followed by a terrestrial lung-breathing adult stage. Amphibians include the class of amphibians and the orders of Anura (frogs or toads) and Urodela (newts or salamanders, and Apoda (cecilians). In embodiments, the amphibians described herein are young or neotenic amphibians. A young amphibian includes a young frog, such as a froglet, tadpole, or larval stage young Apoda. The amphibian can be a young amphibian and the young amphibian can include a larval stage of any order of amphibian. The amphibian can be neotenic.

[0054] The term “salamanders” refers to a group of amphibians characterized by a lizard-like appearance and having a tail throughout life. The families of salamanders include the Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), Proteidae (mudpuppies and olms), Rhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The Ambystomatidae family includes Ambystoma altamirani, Ambystoma amblycephalum, Ambystoma andersoni, Ambystoma annulatum, Ambystoma barbourin, Ambystoma bishop,Ambystoma bombypellum, Ambystoma californiense, Ambystoma cingulated, Ambystoma dumerilii, Ambystoma flavipiperatum, 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, Ambystoma rosaceum, Ambystoma silvense, Ambystoma subsalsum, Ambystoma talpoideum, Ambystoma taylori, Ambystoma texanum, Ambystoma tigrinum, and Ambystoma velasci.

[0055] The families of salamanders are grouped under the order Urodela (or Caudata). The term “Urodele” refers to a salamander of the order Urodela, in the class Amphibia. The salamanders can be from the orders Urodela and Apoda. Urodeles begin life as aquatic animals in a larval state, and some undergo metamorphosis from a juvenile form with gills to an adult, terrestrial, air-breathing form with lungs. During metamorphosis, a Urodele's physical features are altered in preparation for life on land. These alterations include caudal fin resorption, thickening of the skin, the development of dermal glands, and resorption of gills. Sexual maturity also occurs during this time in most Urodeles. However, some families of Urodeles are "neotenic," which means that individuals of such families, even after reaching sexual maturity, retain their juvenile aquatic form throughout their lives. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of neotenic salamanders. Instead of becoming a terrestrial amphibian, an adult axolotl remains aquatic and gilled. However, under certain circumstances, an axolotl will undergo metamorphosis and transform into a terrestrial form.

[0056] Axolotls can fully regenerate lost or damaged body parts including organs, limbs, and parts of the central nervous system, throughout their entire life. Aquatic axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scar-less processes. Similarly, metamorphic terrestrial axolotls, which retain several larval skin features, also exhibit scar-free wound healing, but at a slower rate than their aquatic, pre-metamorphic counterpart. The axolotl wound healing process resembles the scar-free healing process of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than do the corresponding events in postnatal mammals.

[0057] Blastema is a mass of undifferentiated cells that can develop into an organ. In amphibians, the blastema is important in the regeneration of severed limbs. Blastemas are found in the early stages of an organism’s development such as embryos and in the regeneration of tissues, organs, and bone. Some amphibians, such as salamanders, can produce blastemas even as adults allowing them to regenerate their limbs, tail, and other organs after amputation. Ambystoma mexicanum is a neotenic salamander with exceptional regenerative capabilities. Limb regeneration in these salamanders involves blastema.

[0058] Blastemas isolated from amphibians, such as Urodeles, less than 7 days after injury and subsequently activated to induce the release of intracellular components from the blastemas have wound healing potential. Blastemas can be harvested from the site of injury 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days after injury and then activated for treating wounds. In embodiments, the blastemas harvested 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 and subsequently activated have wound healing potential.

[0059] The injury to the amphibian for producing blastemas can include different body parts of the amphibian, such as to the forelimbs including the front legs, feet, and 4 toes; the rear limbs including the rear legs, feet, and 5 toes; the tail; the spinal cord; the law jaw; the brain including telencephalon or forebrain; the heart include the heart ventricle; and portions of the eyes.

[0060] After harvesting the blastema from the injury site, the blastemas can be activated by physical, chemical, and / or electrical disruption involving cell lysis to induce the release of intracellular components from the blastemas. Examples of physical disruption methods include mechanical disruption using an apparatus such as a blender, homogenization using an ultrasonic homogenizer, sonication, freeze-thaw, and manual grinding. Chemical disruption includes using anionic, non-ionic, or zwitterionic detergent to lyse the cells. Examples of such detergents include sodium dodecyl sulfate (SDS, anionic), Triton X-100 (non-ionic, Triton X-114 (non-ionic), NP-40 (non-ionic), Tween 20 (non-ionic), Tween 80 (non-ionic), CHAPS (zwitterionic) and CHAPSO (zwitterionic). Other forms of cell lysis include cytolysis and osmotic lysing. Electrical lysis involves applying a high electric field to the blastemas to break the cell membrane. An example of electric lysis is irreversible electroporation.

[0061] The activated blastemas described herein have enhanced properties. For example, the activated blastemas exhibit enhanced properties at a wound site as compared to a control wound site. The control can be untreated, for example, a wound site that has not been treated with the activated blastemas. The control can also be a wound site that has been treated with activated blastemas prepared from blastemas harvested more than 7 days after injury. The control can be a wound site treated with blastemas that have not been activated.

[0062] The present application describes compositions comprising activated amphibian blastemas. The activated blastemas are a mixture of amphibian blastema cells that have been disrupted to release intracellular components from the blastemas. The activated amphibian blastemas can be obtained from various injured amphibians, such as Urodeles, described herein. In embodiments, the amphibian blastemas are obtained from injured Ambystoma mexicanum.

[0063] The composition described herein can include carriers and / or excipients. Examples of carriers and excipients include saline, emulsion, a mixture of organic solvents with water, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, petrolatum, lanolin, mineral oil, dimethicone, humectant, and polyethylene glycols. Examples of humectants include glycerin, lecithin, and propylene glycol. In embodiments, thecompositions described herein include cosmetic, or pharmaceutical compositions containing one or more cosmetically, or pharmaceutically acceptable carriers or excipients, respectively.

[0064] The compositions described herein can include carriers for immediate or sustained release preparations. Such carriers include polymers. The polymers can be biodegradable, and / or bioabsorbable. As an example, for controlled release, the biomaterial can be coated with polymers such as acrylic polymer, acrylic / methacrylic copolymer, cellulose acetate phthalate (CAP), Opadry®, and Ethocel™. For immediate release, the biomaterial can be coated with cellulosic polymers, such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethylcellulose (HEC), methylcellulose (MC), and sodium carboxymethyl cellulose (NaCMC); vinyl derivatives, such as polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol (PVA), and polyvinyl alcohol-polyethylene glycol copolymer; acrylic polymers, such as Eudragit®; or glycols such as polyethylene glycols.

[0065] The compositions described herein can include one or more agents, such as therapeutic agents or cosmetic agents. Examples of therapeutic agents include known drugs such as retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics. Examples of cosmetic agents include antioxidants, peptides, alpha or beta hydroxy acids, retinol, vitamins, plant extracts, skin clarifying agents such as arbutin, moisturizing agents such as hyaluronic acid, emollients, carbohydrates, glycoproteins, and / or polymers. The one or more agents can include a combination of agents. The agent can be exogenous or xenogenic to the activated blastema.

[0066] 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, neurotrophin- 5; platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); stromal- 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.

[0067] Examples of one or more therapeutic agents include antimicrobials and anti- inflammatory agents. Examples of antimicrobials include antibiotics such as penicillin, streptomycin, amoxicillin, cephalexin, clindamycin, dicloxacillin, and doxycycline. Other antimicrobials include anti-microbial peptides, silver salts, clotrimazole, miconazole, andketoconazole. 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.

[0068] 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 absorbing agents, 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.

[0069] The compositions described herein can also include one or more natural and / or synthetic polymers. Natural polymers can be from an animal source or a non-animal source such as a plant source. 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 acids), polydioxanone, polycaprolactone, poly(ortho esters), poly(anhydrides), polyphosphazene, poly(amino acids), pseudo-poly(amino acids), conductive polymers (such as polyacetylene, polypyrrole, polyaniline), polyurethane, polystyrene, and nitinol.

[0070] The polymer of the compositions described herein can be biocompatible, biodegradable, and / or bioabsorbable, and can be a random copolymer, block copolymer, or blend of monomers, homopolymers, copolymers, and / or heteropolymers that contain these monomers. Exemplary biodegradable or bioabsorbable polymers include polylactides, poly-glycolides, polycaprolactones, polydioxanes, and their random and block copolymers. A biodegradable and / or bioabsorbable polymer can contain a monomer selected from the group consisting of glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol, and lysine. The biodegradable and / or bioabsorbable polymers can contain bioabsorbable and biodegradable linear aliphatic polyesters such as polyglycolide (PGA) and its random copolymer poly(glycolide- co-lactide-) (PGA-co-PLA). Other examples of suitable biocompatible polymers include polyhydroxyalkyl methacrylate, ethylmethacrylate, polyvinylpyrrolidone, and polyacrylamides. Other suitable bioabsorbable materials are biopolymers which include collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acid, polylysine, and copolymers of these materials. Any combination of polymers and copolymers or blend thereof of the above examples can also be included in the composition.

[0071] The compositions described herein can also include protectives, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffering agents, solubilizing agents, skin-penetration enhancers, and surfactants.

[0072] A skin penetration enhancer can be added to the compositions described herein, provided the skin penetration enhancer is safe and can effectively facilitate the passage of the desired substances in the activated blastemas across the skin membrane. Examples of skin penetration enhancers include dimethyl sulphoxide (DMSO), monoglycerides, C10-C20 fatty acid esters including ethyl palmitate and isopropyl myristate; acyl lactylates such as caproyl lactylic acid and lauroyl lactylic acid; dimethyl lauramide; dodecyl (lauryl) acetate; lactate esters such as lauryl lactate, and myristyl lactate; monoalkyl ethers of polyethyleneglycol and their alkyl or aryl carboxylic acid 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-azepine 2-one (Azone), oleic acid, linoleic acid, isopropyl linoleate, oleyl alcohol, 1-dodecyl-azacycloheptan-2-one, butanediol, and 2-(2-Ethoxyethoxy)ethanol (Transcutol).

[0073] The compositions described herein can be in the form of a dry powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, a gel, a hydrogel, a gelatin, or an emulsion.

[0074] The compositions described herein also can 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 any scarring. The compositions described herein can reduce inflammation, reduce scarring, reduce keloid formation, reduce or diminish the severity of scarring and keloid formation, and / or reduce healing time for various dermatological and cosmetic procedures. The compositions described herein can also be used to restore lost dermal matrix or subdermal volume.

[0075] Skin diseases or conditions include inflammatory and cancerous skin conditions. Examples of skin conditions requiring treatment include acne, actinic keratosis, blister, cellulitis, cold blister, hives, impetigo, Keratosis pilaris, melasma, moles, ringworm, urticaria, vitiligo, and wart. Examples of inflammatory skin conditions include psoriasis; dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, stasis dermatitis, 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 needing treatment include fine lines and / or wrinkles, aging, redness, abrasion, burn, cut, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The burns include acute thermal burns such as first, second,or third-degree burns. Skin conditions also include wounds including third-degree burns, deep dermal wounds, and full thickness wounds.

[0076] The compositions described herein can be administered to the target site topically, or by injection, implantation, microneedling, or radiofrequency microneedling, or using an ablative fractional laser. The isolated compositions described herein can be administered to the subject prior to, during, or after a dermatological or cosmetic procedure, such as dermabrasion, microdermabrasion, and ablative laser resurfacing. The dermatological or cosmetic procedure includes procedures wherein at least one cell of the stratum corneum is removed. The 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 including collagen or elastin, either naturally occurring, bioengineered, or recombinantly produced. The 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 compositions can also be administered with toxins, such as botulinum toxin.

[0077] The compositions described herein can be applied topically, for example directly to the wound or skin, or indirectly, by applying it to a substrate that is used to cover the wound or the skin. The compositions can also be applied to a device, such as a medical device, for administration to the subject.

[0078] Prior to treatment with activated blastemas, the wound site can be treated using a laser, micro-coring, microneedling, or using any dermatological or cosmetic procedures wherein at least one cell of the stratum corneum is removed. These procedures can assist in the delivery of the activated blastemas and enhance wound healing without scarring. Treatment with a laser includes treating the wound with an ablative fractional laser. Microneedling includes treating the wound with radiofrequency microneedling.

[0079] In embodiments, the compositions described herein can enhance the epithelial thickness of the wound providing a robust skin at the wound site. The compositions can also increase the re-epithelialization rate which indicates that they can accelerate the wound healing process. Moreover, the compositions exhibited wound closure efficiency, especially for third- degree burns as demonstrated by the 100% re-epithelialization by Day 14 with laser treatment. Further, the compositions can decrease white cell infiltration which indicates that the compositions can minimize scarring and faster recovery.

[0080] The present disclosure describes kits including the compositions described herein for the various uses described herein. The kits can include sterilized compositions in any shape and form. The kits can include a solution for reconstituting the compositions for use. The kits can include a device for administering the composition to a subject. The kits can include an implant to be coated with the composition prior to being implanted in a subject. The kits can include components for the various uses described herein.

[0081] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0082] Numbers expressing ranges or quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ± 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 ± any percentage between 1% and 20% of the stated value.

[0083] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. In embodiments, the lack of a material effect of a step is evidenced by the lack of a statistically significant wound healing as determined by one or more of the parameters described herein, for example, the percentage of wound epithelialized, epithelial thickness, granulation tissue formation, or planimetry measurements (wound area). Lack of a material effect of an embodiment can include the lack of a statistically significant improvement in wound healing as determined by one or more of the parameters described herein.

[0084] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc.,as well as individual numbers within that range, for example, 1, 2, 2.5, 2.7, 3, 4, 5, 5.1, 5.3, 5.8 and 6. Moreover, any ranges cited herein are inclusive.

[0085] 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 and claimed individually or in any combination with other members of the group or other elements found herein.

[0086] The following embodiments and examples illustrate exemplary methods provided herein. These examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure. EXEMPLARY EMBODIMENTS

[0087] The following are exemplary embodiments. 1. A composition including activated amphibian blastemas, wherein the activated amphibian blastemas include enhanced properties as compared to a control, and wherein the blastemas are obtained from an injured amphibian less than 7 days after injury, and optionally wherein the composition includes a carrier. 2. The composition of embodiment 1, wherein the control includes blastemas obtained from an amphibian that is not activated or wherein the control includes blastemas obtained from an amphibian 7 or more days after injury. 3. The composition of embodiment 1 or 2, wherein the composition includes a homogenized mixture of amphibian blastemas. 4. The composition of any one of embodiments 1-3, wherein the amphibian is a frog, toad, newt, or salamander. 5. The composition of any one of embodiments 1-4, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian includes a froglet, tadpole, Urodele, or larval stage young Apoda. 6. The composition of any one of embodiments 1-5, wherein the amphibian is a neotenic Urodele. 7. The composition of any one of embodiments 1-6, wherein the activated amphibian blastemas are obtained from the injury site between 12 hours to 7 days after injury, from 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days, or 3 days after injury. 8. The composition of any one of embodiments 1-7, wherein the composition is a pharmaceutical composition or cosmetic composition further including a pharmaceutically acceptable carrier or cosmetically acceptable carrier.9. The composition of any one of embodiments 1-8, wherein the composition further includes one or more agents xenogenic to the activated blastemas. 10. The composition of any one of embodiments 1-9, wherein the composition further includes one or more agents including peptides, proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof; and optionally wherein the one or more proteins or peptides include growth factors, cytokines, or chemokines; optionally wherein the one or more polymers includes synthetic or natural polymers or copolymers; optionally wherein the one or more drugs include retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics; and optionally wherein the one or more drugs include neomycin, polymyxin B, bacitracin, or a combination thereof. 11. The composition of any one of embodiments 1-10, wherein the composition is in the form of a gel, a paste, a solution, a serum, a cream, a lotion, dispersion, or an emulsion. 12. A method of preparing a composition including activated amphibian blastemas, wherein the method includes injuring the amphibian, allowing the amphibian to recover from the injury, isolating blastemas from an injury site of the amphibian less than 7 days after the injury, and activating the isolated blastemas to induce release of intracellular components from the blastemas, thereby preparing a composition including activated amphibian blastemas. 13. The method of embodiment 12, wherein activating the isolated blastemas includes mechanical, chemical, and / or electrical disruption causing cell lysis to induce release of intracellular components from the blastemas. 14. The method of embodiment 12 or 13, wherein activating the isolated blastemas includes mechanical disruption including using a blender, a homogenizer, a sonicator, or a freeze-thawing process, or manually grinding the isolated blastemas and optionally wherein the homogenizer is an ultrasonic homogenizer. 15. The method of any one of embodiments 12-14, where activating the isolated blastemas includes chemical disruption including using anionic, non-ionic, or zwitterionic detergent to lyse the cells. 16. The method of any one of embodiments 12-15, wherein activating the isolated blastemas includes using one or more detergents including sodium dodecyl sulfate (SDS, anionic), Triton X- 100 (non-ionic, Triton X-114 (non-ionic), NP-40 (non-ionic), Tween 20 (non-ionic), Tween 80 (non-ionic), CHAPS (zwitterionic) and CHAPSO (zwitterionic). 17. The method of any one of embodiments 12-16, wherein activating the isolated blastemas includes electrical disruption including irreversible electroporation. 18. The method of any one of embodiments 12-17, wherein activating the isolated blastemas includes cytolysis or osmotic lysing.19. The method of any one of embodiments 12-18, wherein activating the isolated blastema includes lysing the cells in saline solution, and optionally wherein the saline is phosphate buffered solution or physiological saline solution. 20. The method of any one of embodiments 12-19, wherein the amphibian is a frog, toad, newt, or salamander. 21. The method of any one of embodiments 12-20, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian includes a froglet, tadpole, Urodele, or larval stage young Apoda. 22. The method of any one of embodiments 12-21, wherein the amphibian is a neotenic Urodele. 23. The method of any one of embodiments 12-22, wherein the method includes isolating blastemas from the injury site between 12 hours to 7 days after injury, from 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days, or 3 days after injury 24. The method of any one of embodiments 12-23, wherein injuring the amphibian includes any method that induces blastema formation. 25. The method of any one of embodiments 12-24, wherein injuring the amphibian includes amputating a limb or tail or causing a wound and optionally wherein the wound is a skin wound. 26. The method of any one of embodiments 12-25, wherein the method further includes preparing the activated blastema into a gel, apaste, a solution, a serum, an extract, a cream, a lotion, a dispersion, a powder, or an emulsion. 27. A method of treating or preventing a skin disease or condition in a subject in need thereof including administering the composition of the activated amphibian blastema of any one of embodiments 1-11 or the activated amphibian blastema obtained by the method of any one of embodiments 12-26 to the subject. 28. The method of embodiment 27, wherein the skin condition includes fine lines and / or wrinkles, aging, redness, abrasion, burns, blisters, cuts, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, increasing skin elasticity and / or firmness, improving skin hydration, inflammation, or hyperpigmentation. 29. The method of embodiment 27 or 28, wherein preventing a skin condition includes protecting the skin from damages caused by UV rays and / or environmental pollution. 30. The method of any one of embodiments 27-29, wherein the method further includes treating the skin with a laser before administering the activated blastema, and optionally wherein the laser is ablative fractional laser. 31. The method of any one of embodiments 27-30, wherein the skin condition is a burn including a first, second, or third-degree burn. 32. The method of any one of embodiments 27 to 30, wherein the skin condition is a wound including a deep dermal wound or a full thickness wound.33. The method of any one of embodiments 27-32, wherein the skin condition is a wound and wherein the treatment with the composition enhanced epithelial thickness of the skin at the wound site, increased re-epithelialization rate, increased wound healing process, increased wound closure efficiency, decreased white cell infiltration, and / or decreased scarring. EXAMPLES

[0088] Regeneration, the remarkable ability of organisms to replace lost or damaged tissues, has long captivated the imagination of scientists and the public alike. Among nature's proficient regenerators, the axolotl (Ambystoma mexicanum) stands out as an extraordinary model organism due to its unparalleled regenerative prowess. From regrowing entire limbs to reconstructing complex organs, the axolotl has intrigued researchers for centuries.

[0089] Wound healing is a complex, multi-phase process that involves coordinated interactions between various cell types, extracellular matrix components, and signaling molecules. Despite significant advancements in medical science, chronic and non-healing wounds remain a substantial clinical challenge, affecting millions of people worldwide and imposing a significant burden on healthcare systems. Current treatments often fail to achieve complete and functional tissue regeneration, highlighting the need for innovative therapeutic strategies.

[0090] This study presents the results of using an axolotl-derived gel on third-degree burns, deep dermal wounds, and full thickness wounds in a wound-healing pig model. This study integrates insights from comparative biology, developmental genetics, and tissue engineering to explore the therapeutic potential of axolotl for human tissue regeneration. Moreover, the investigators involved in this study opted to use an in-vivo model, in particular a pig wound healing model for several reasons. Anatomically and physiologically, pig skin is remarkably similar to human skin, making it an ideal model for experimental studies. Both pig and human skins feature an epidermis ranging from 50 to 120 microns. Given the variability in epidermal thickness across different anatomical regions, a more precise comparison is provided by the dermal-to-epidermal thickness ratio. In both species, this ratio is consistently approximately 10:1 to 13:1, underscoring the close resemblance. Furthermore, both pig and human skins exhibit well-defined rete ridges and dermal papillary bodies, as well as abundant subdermal adipose tissue. The biochemical similarity between porcine and human dermal collagen further supports the use of pig skin in developing wound healing models highlighting its relevance in translational research and therapeutic applications. Humans and pigs heal through physiologically similar processes. Most small animals have a panniculus carnosus and rely on wound contraction for wound closure. Conversely, man and swine close wounds largely through re-epithelialization. Additionally, the pig’s overall physiology is close to human physiology, with most key organ systems being similar in anatomy and function.

[0091] Example 1. Preparation of Activated Blastemas

[0092] Axolotls of different phenotypes were used in this study. The animals were maintained at 20–22°C in dechlorinated water. For surgical procedures, the axolotls were anesthetized using 0.1% ethyl 3-aminobenzoate methane sulfonate salt (Sigma, MS222) and adjusted to pH 7.0. All limbs were amputated below the knee for hindlimbs or below the elbow for forelimbs using a sterile scalpel (No.10).

[0093] All procedures were conducted under the guidance of a licensed veterinarian, ensuring the animals were adequately sedated to minimize discomfort and comply with ethical standards for the use of animals in research.

[0094] On day 0, axolotls were injured by trimming their front right and back left limbs. Blastemas were harvested from the site of injury on day 1, day 2, day 3, day 4, and day 7 and subsequently activated for treating wounds. The harvested Blastemas were sonicated according to the protocol outlined in Table 1. Sonication was performed while the samples were kept cool in an ice bath to minimize thermal degradation. After sonication, the resulting gel was transferred to a new vial and refrigerated until use.

[0095] Table 1: Sonication Procedure Parameter Value Process Time 1 minute

[0096] Treatment preparaton was conducted mmedatey pror to treatment admnstraton on the pig wound model (described below) on the initial day of the experiment (Day 0). Subsequent doses were derived from the original preparation. It is important to note that the treatment material was stored at 2-8°C. This ensures that the material used in all treatments was consistent with the initial preparation, maintaining the integrity of the experimental conditions.

[0097] Gel Z is activated blastema harvested from axolotl one day after injury. Gel T is activated blastema harvested from axolotl two days after injury. Gel Y is activated blastema harvested from axolotl three days after injury. Gel F is activated blastema harvested from axolotl four days after injury. Gel X is activated blastema harvested from axolotl seven days after injury.

[0098] The phrase “activated blastema Day #” refers to activated blastemas prepared from blastema harvested on Day # after injury. # number such as 1, 2, 3, 4, or 7.

[0099] Example 2. Animal Studies

[0100] The objective of these studies is to determine the ability of activated blastemas to enhance healing using three different porcine wound models.

[0101] Experimental Animals. Porcine models were used for the experimental research due to the morphological similarities between swine skin and human skin. Two (2) female animals’ specific pathogen-free (sourced from B. G. Looper Farm 4673 Petra Mill Road Granite Falls, NC 28630) pigs weighing 40-45 kg were kept in-house for at least 5 days prior to the commencement of the experiment to allow for acclimatization. One animal received third-degree burns and the other deep dermal wounds as described below. The animals were fed a basal diet ad libitum and housed individually in our animal facilities (meeting American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature (19-21oC) and lighting (12h / 12h LD).

[0102] Wound Models

[0103] The wound models include the third-degree burn model, deep dermal wound model, and 20 mm full thickness wound model. Trials A and B were performed using these models.

[0104] Third-Degree Burn Model. FIG.1 shows an exemplary study design for third-degree burn wounds. Twenty-five (25) third-degree burn wounds were made on the paravertebral and thoracic areas. Burn wounds were created by using a branding iron (L & H Manufacturing Company Mandan, North Dakota 58554) with a heat controller that was set to 300oC. The iron was held at a vertical position on the skin for 15 seconds, with pressure supplied by gravity, to make a burn wound of 27mm in diameter and with a depth of approximately 3mm (to subcutaneous tissue). The wounds were separated from one another by 5-7 cm of unwounded skin. The wounds were randomly assigned to six treatment groups with 3 or 4 wounds per treatment as seen in FIG.1. One of three wounds was first lasered prior to treatment. The animal was treated within 20 minutes after the creation of the wound.

[0105] Deep Dermal Wound Model. FIG.2 shows an exemplary study design for deep dermal wounds. Deep reticular dermal wounds measuring (22 mm x 22 mm x 3 mm, L x W x D) were made in the paravertebral and thoracic area with a specialized electrokeratome fitted with a 22 mm blade. The wounds were separated from one another by 5-7 cm of unwounded skin. The wounds were randomly assigned to six treatment groups with 3 or 4 wounds per treatment as seen in FIG.2. One of three wounds was first lasered prior to treatment. The animal was treated within 20 minutes after the creation of the wound.

[0106] 20 mm Full Thickness Wound Model. FIG.3 shows an exemplary study design for full thickness wounds. Full thickness wounds were made on the paravertebral and thoracic area with a 20mm circular biopsy punch (FIG.3). The wounds were separated from one another by 5-7 cm of unwounded skin. The wounds were randomly assigned to seven treatment groups with 3 wounds per treatment as seen in FIG.3. The animal was treated within 20 minutes after creation of the wound.

[0107] Treatment Regimen

[0108] Within the first hour after wounding, some of the wounds were treated with an Erbium YAG fractional ablative laser. This laser creates numerous microscopic columns of tissue ablation, enhancing the delivery and penetration of topical agents into the wound bed. The columns act as channels to enhance the delivery of topical agents, allowing for better penetration into the wounded area. The laser was set at 15 mJ for micropulse energy at a rate of 300 Hz and a repeat delay of 30 seconds.

[0109] Immediately after lasering, all the third-degree burn wounds and deep dermal wounds received 200 μL of the gel formulation of activated blastemas (prepared from blastemas harvested on Days 1, 2, 3, 4, and 7) after treatment that was spread with a sterile spatula to cover the wounded area and surrounding unwounded skin and covered with Tegaderm dressing. All the 20 mm full thickness wounds received 100 μL of the gel formulation of activated blastemas (prepared from blastemas harvested on Days 1, 3, and 7 after injury). Untreated Control wounds for the animals were covered with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN). After application, all treatment groups were covered with Tegaderm.

[0110] All wounds were treated for 7 days (from initial wounding Day 0 until Day 6). All wounds were covered after treatment application with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN). After the initial 7 days, wounds were covered with non-adherent gauze until the final assessment. On Day 6 (7 days post-wounding), all wounds received a final application of the treatment and were subsequently dressed with non-adherent gauze and Tegaderm. All dressings were secured with tape and covered with Coban wrap (3M, St. Paul, MN).

[0111] Assessments

[0112] Histological Assessment. On assessment days (Day 14 for third-degree burns and Day 10 for deep dermal wounds and full thickness wounds), incisional biopsies were taken from each treatment group using a sterile scalpel. Biopsies were obtained through the center of the wounds including normal adjacent skin on both sides. These specimens were placed in formalin and then stained with hematoxylin and eosin (H&E). One section per block was analyzed. The specimens were then scanned on an Olympus VS120 slide scanner and blindly evaluated for the following parameters to determine a potential treatment response: Percent of wound epithelialized (%). Measurement of the length of the wound surface that has been covered with epithelium. Epithelial thickness (cell layers μm). The epithelial thickness may vary within the biopsy. The thickness of the epithelium in micrometers (μm) was measured on five equal distance points from each other in the biopsy and averaged. White cell infiltrate. The presence and amount of subepithelial mixed leukocytic infiltrates were measured. The mean score was assigned as follows:: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, and 5 = exuberant.Granulation Tissue Formation. The approximate amount of new granulation tissue formation in the dermis was graded as follows: 0 = 0, 0.5 = 1-10%, 1 = 11-30%, 2 = 31- 50%, 3 = 51-70%, 4 = 71-90%, and 5= 91-100%.

[0113] This comprehensive analysis provided crucial insights into the efficacy of the treatments in promoting wound healing, epithelial regeneration, and inflammatory response.

[0114] Histological Results.

[0115] The data were combined and analysis was performed to determine the treatment response. The percentage of epithelialization and the other histological parameters in wounds were plotted against days after treatment. Assessments for third-degree burn wounds, deep dermal wounds, and full thickness wounds were on Days 14, 10, and 10 respectively. Since there were only one, two, or three samples for each treatment group, only means were used for tabulation and graphs for presentation purposes.

[0116] Percentage of Epithelialization. The percentage of epithelialization represents the area of the wound area covered by a newly formed epidermis with one or more layers of keratinocytes, which is a good index for the speed of keratinocyte migration and the first step of the epithelialization. The percentages of epithelialization for the different trials using the three different wound models treated with activated blastemas prepared from blastemas harvested on Days 1, 2, 3, 4, and 7 after injury, as compared to control are shown in FIGs.4-14. The results show that treatment with activated blastemas prepared from blastemas harvested less than 7 days after injury to the axolotl and subsequently activated can be used with or without laser pretreatment to effectively treat different wounds

[0117] Epithelial thickness. The epithelial thickness was a measure of an average thickness of five points of newly formed epithelium. Epithelial thickness reflects the process of keratinocyte proliferation, differentiation, and epidermal maturation (FIGs 21 and 22).

[0118] White Cell Infiltration (WCI). WCI is used to assess the inflammation reaction (FIGs. 15, 16, 23, and 24).

[0119] Granulation Tissue Formation. The dermal reconstitution begins within about 3 to 4 days of injury with the hallmark of granulation tissue formation, which includes new blood vessel formation (angiogenesis), accumulation of fibroblasts, and collagen extracellular matrices. The granulation tissue formation measures the percentage of wound beds filled with newly formed granulation tissue (FIGs 17, 18, 25, and 26).

[0120] Planimetry Measurements

[0121] The wound areas were combined for each treatment group at each time point (every 3- 4 days) and were analyzed for comparative quantitative analysis. A comparison was made of the wound area of different treatments using activated blastemas (prepared from blastemas harvested on Days 1, 2, 3, 4, and 7 after injury) applied after laser application for third-degreeburn wounds. At Day 14, the wound area is lower than the untreated control confirming that the activated blastemas are effective in treating wounds (FIGs 27-30).

[0122] Clinical Observations (Erythema).

[0123] Erythema – indicative of the amount of inflammation present*

[0124] ∗ Score: 1 = absent, 2 = mild, 3 = moderate, 4 = marked, 5 = exuberant

[0125] All animals exhibited no erythema (absent score) on all wounds throughout the study.

[0126] Digital Photography & Measurement of the Wound Contraction. Wounds from each group were photographed, and the wound area was traced to measure wound contraction. The wound circumference was meticulously outlined using digital imaging with ImageJ, a software tool developed by the National Institutes of Health (NIH). ImageJ is widely used in the scientific community for image analysis due to its powerful features and user-friendly interface.

[0127] To assess wound contraction, the circumference of each wound was traced and compared to the measurements taken on Day 0. This comparison allowed for precise quantification of the degree of wound contraction over time. ImageJ's capabilities in handling large datasets and its advanced image processing algorithms made it an ideal choice for this analysis, ensuring accuracy and reproducibility in our measurements.

[0128] Results

[0129] Third-Degree Burn Wounds. Third-degree burns treated with activated blastema Day 3 group, without the supplementation of laser treatment, achieved 100% re-epithelialization by Day 14. Notably, this was the only treatment modality to reach complete re-epithelialization within the study period. In contrast, the untreated control group without laser exposure reached 76.20% re-epithelialization over the same duration. Among the non-lasered groups, the activated blastema Day 1 group (47.95%) resulted in the lowest re-epithelialization rate by Day 14 closely followed by the activated blastema Day 7 group (48.3%, FIG.9A1). Nonetheless, these two latter groups increased by greater than 50% in re-epithelialization when laser treatment was incorporated as part of the treatment (FIG.9A2). The opposite is true for activated blastema Days 2 and 3 groups, where the supplementation of laser treatment had an antagonistic effect.

[0130] The results indicate that the epithelial thickness in third-degree burns at Day 14 without laser treatment varied depending on the timing of the test article (activated blastema), i.e., Day 1, 2, 3, 4, or 7 post blastema induction. The activated blastema Day 2 group had the highest epithelial thickness at 122.1 µm, followed by the activated blastema Day 7 group with a thickness of 118.1 µm (FIG.13). The activated blastema Day 1 group showed a thickness of 99.75 µm, while the activated blastema Day 3 and Day 4 groups had thicknesses of 81.35 µm and 82.5 µm, respectively. The untreated control group exhibited an epithelial thickness of 87.95 µm.

[0131] Epithelial thickness in groups supplemented with laser treatment prior to treatment with activated blastema resulted in a tighter distribution. Activated blastema Day 4 group showed the thickest epithelium by Day 14, with 112.8 µm (FIG.14). This was closely followed by the untreatedcontrol and activated blastema Day 2 group, with measurements of 110.7 µm and 107.9 µm, respectively. Activated blastema Day 1 group yielded the thinnest epithelium measurement at 76.5 µm.

[0132] Activated blastema Day 7 group showed the highest score for white cell infiltrate, 3.5, followed by activated blastema Day 1 group with a score of 3 on non-lasered wounds (FIG.15). Both activated blastema Day 3 group and untreated control group, without laser, yielded the lowest score at 2. On the other hand, activated blastema treatments that were supplemented with laser treatment showed a decrease in white cell infiltrate score except for the activated blastema Day 2 and Day 3 groups, both scoring 3 (FIG.16).

[0133] Granulation tissue scores exhibited a similar trend to white cell infiltration scores when comparing treatment modalities with and without laser supplementation. Specifically, activated blastema Day 2 and Day 3 groups without laser resulted in the lowest granulation tissue score of 2.5, followed closely by the untreated control group without laser at 3 (FIG. 17). In contrast, activated blastema Day 4 and Day 7 groups without laser supplementation consistently achieved granulation tissue scores of 3.5 (FIG. 17). Notably, when laser treatment was introduced, the granulation tissue scores for activated blastema Day 2 and Day 3 treatments increased significantly to 4, representing a marked increase of 1.5 points (FIG. 18). However, the granulation tissue score for activated blastema Day 7 treatment decreased to 2 when laser treatment was included (FIG.18).

[0134] Deep Dermal Wounds. Similar to the findings in the third-degree wound model, activated blastema Day 3 group administered without laser resulted in 100% re-epithelialization by Day 10 in deep dermal wounds (FIG. 19), followed by activated blastema Day 2 group at 86.1%, activated blastema Day 4 group at 85.4%, untreated control group at 78%, activated blastema Day 7 group at 70.6%, and activated blastema Day 1 at 60.9% (FIG. 19). The supplementation of laser prior to activated blastema treatment yielded different results. For wounds that received laser treatment, the results show 100% re-epithelialization for activated blastema Day 1 group, 95.8% for untreated control, 90.8% for activated blastema Day 3 group, 85.5% for activated blastema Day 4 group, 63.1% for activated blastema Day 7 group, and 62.4% for activated blastema Day 2 group (FIG.20).

[0135] The epithelial thickness in deep dermal wounds at Day 10 post-injury, without laser treatment, varied across the different activated blastema treatments. Activated blastema Day 4 group exhibited the highest epithelial thickness, measuring 127.7 µm (FIG. 21), followed by activated blastema Day 1 group, which showed an epithelial thickness of 117 µm (FIG. 21). Activated blastema Day 7 and Day 3 groups exhibited thicknesses of 105.65 µm and 100.35 µm, respectively (FIG. 21). The untreated control group without laser treatment had an epithelial thickness of 97.4 µm (FIG. 16). The lowest epithelial thickness was observed in the activated blastema Day 2 group, with a measurement of 78.2 µm (FIG.21).

[0136] On the other hand, with laser treatment, the epithelial thickness of deep dermal wounds at Day 10 post-injury showed that activated blastema Day 7 group had the highest epithelial thickness, measuring 157.4 µm, followed by activated blastema Day 2 group with an epithelial thickness of 140.3 µm (FIG.22). The untreated control group with laser treatment had a thickness of 93.4 µm (FIG.22). Activated blastema Day 3 and Day 1 group exhibited an epithelial thickness of 90.6 µm and 77.7 µm, respectively (FIG.22). The lowest epithelial thickness was observed in the activated blastema Day 4 group, with a measurement of 65.7 µm.

[0137] The white cell infiltrate (WCI) in deep dermal wounds at Day 10 post-injury, without laser treatment, showed that activated blastema Day 1 group presented the highest WCI score of 4 (FIG. 23), indicating a significant inflammatory response. In contrast, all the other activated blastema treatments – activated blastema Days 2, 3, 4, and 7 groups -- had a consistent WCI score of 2 (FIG. 23),. The untreated control group without laser treatment exhibited the lowest WCI score of 1.5 (FIG. 23),. For lasered treatments, all activated blastema treated groups received a score of 1, while untreated lasered control received a score of 3, double what was documented for untreated control without laser (FIG.24),.

[0138] The granulation tissue formation in deep dermal wounds at Day 10 post-injury, without laser treatment, showed that activated blastema Day 1 group and Day 2 group both showed the highest granulation tissue score of 2 (FIG.25). Activated blastema Day 4 and Day 7 groups, each had a granulation tissue score of 1.5, while activated blastema Day 3 group and the untreated control group showed the lowest granulation tissue score of 1 (FIG.25). Deep dermal wounds that received laser treatment before activated blastema treatment, showed consistency across most of the activated blastema treatment groups. Activated blastema Day 1, Day 2, Day 4, and Day 7 groups all resulted in the highest granulation tissue score of 2 (FIG.26). In contrast, the untreated lasered group and activated blastema Day 3 lasered group presented a lower granulation tissue score of 1 (FIG.26).

[0139] Discussion

[0140] The findings from this study elucidate the significant potential of Urodele blastema products, such as axolotl blastema products, in enhancing wound healing, particularly in challenging cases such as third-degree burns and deep dermal wounds in a porcine model. This research offers critical insights into the benefits and mechanisms of using such regenerative therapies, specifically focusing on re-epithelialization, epithelial thickness, white cell infiltration, and granulation tissue formation.

[0141] The results showed a distinct advantage in re-epithelialization when using the activated blastemas. Notably, the third-degree burn wounds treated with the activated blastemas exhibited a re-epithelialization rate of up to 100% by Day 14 post-injury (third-degree Burn), which was markedly higher compared to untreated controls. Similar results were observed in deep dermal wounds albeit less pronounced. This suggests that the combination ofthe activated blastema with laser treatment can significantly accelerate the wound healing process on third-degree burns and possibly deep dermal and full thickness wounds. Furthermore, the results obtained from activated blastema harvested at different time points post-blastema induction (post injury) provide evidence of the importance and complexity of timely harvesting and administering to the subject in regenerative therapies.

[0142] Regarding re-epithelialization, the activated blastema treatment groups, especially the activated blastema Day 3 group, showed remarkable wound closure efficiency. Achieving 100% re-epithelialization in third-degree burns by Day 14 highlights the activated blastema’s capability to enhance skin regeneration significantly. The non-laser treated wounds underperformed in comparison, illustrating the unique regenerative potential of the activated blastemas.

[0143] Epithelial thickness showed variability was observed across treatment timelines, with activated blastema Day 2 group demonstrating the thickest new epithelium. The introduction of laser treatment modified epithelial outcomes, often augmenting thickness, particularly with Day 7 samples. These variations underline the importance of timing and sequencing in regenerative protocols.

[0144] Epithelial thickness is a critical marker for the quality and robustness of the healed skin. The results revealed that non-lasered treatments generally led to a thicker epithelial layer compared to their lasered counterparts. For instance, with the third-degree burn, the activated blastema Day 2 group with no laser group achieved an epithelial thickness of 122.1 µm, which was superior to the 76.5 µm observed in the activated blastema Day 1 lasered group. Similar trends were observed in deep dermal wounds.

[0145] White Cell Infiltration was decreased in certain combinations which led to improved overall wound healing. The reduced inflammatory response in certain lasered groups, as evidenced by lower white cell infiltration scores, points to a potential anti-inflammatory role of laser and axolotl treatment combinations. This aspect is crucial for minimizing scarring and enhancing overall healing quality.

[0146] The non-lasered wounds seem to allow the treatment to concentrate more effectively on the superficial skin layers, rather than penetrating the deeper tissues which is facilitated by the microchannels created during lasering.

[0147] Granulation tissue formation, which is essential for successful wound healing as it provides the scaffold for new tissue growth, was relatively consistent across the treatment groups, with slight variations depending on the treatment approach. Interestingly, non-lasered wounds generally showed better granulation tissue formation, which could be attributed to the less aggressive nature of the treatment, allowing for a more natural healing process. Finally, the study demonstrated that wounds treated with activated blastemas, particularly those combined with laser treatment, exhibited reduced white cell infiltration. For example, the activatedblastema Day 1 lasered group had a white cell infiltration score of 2, compared to 3 in the untreated no laser group. This reduction in inflammation suggests that the activated blastema can help modulate the immune response, potentially leading to less scarring and faster recovery.

[0148] In conclusion, the axolotl-derived activated blastema products, particularly when combined with laser treatment, show great promise in enhancing the healing of third-degree burns, deep dermal wounds, and full thickness wounds. The significant improvements in re- epithelialization and reduced inflammation indicate that this approach could be a valuable addition to current wound care practices, offering new hope for patients with severe and problematic wounds.

[0149] The subject matter described above 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 example embodiments and applications illustrated and described, and without departing from the true spirit and scope of the present disclosure, which is set forth in the following claims.

[0150] All publications, patents, and patent applications cited 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. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. REFERENCES Demircan et al. J. Exp. Clin. Med.2016, 33(4): 229-237 WO 2020 / 032902

Claims

CLAIMS 1. A composition comprising activated amphibian blastemas, wherein the activated amphibian blastemas comprise enhanced properties as compared to a control, and wherein the blastemas are obtained from an injured amphibian less than 7 days after injury, and optionally wherein the composition comprises a carrier.

2. The composition of claim 1, wherein the control comprises blastemas obtained from an amphibian that is not activated or wherein the control comprises blastemas obtained from an amphibian 7 or more days after injury.

3. The composition of claim 1, wherein the composition comprises a homogenized mixture of amphibian blastemas.

4. The composition of claim 1, wherein the amphibian is a frog, toad, newt, or salamander.

5. The composition of claim 1, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian comprises a froglet, tadpole, Urodele, or larval stage young Apoda.

6. The composition of claim 1, wherein the amphibian is a neotenic Urodele.

7. The composition of claim 1, wherein the activated amphibian blastemas are obtained from the injury site between 12 hours to 7 days after injury, from 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days, or 3 days after injury.

8. The composition of claim 1, wherein the composition is a pharmaceutical composition or cosmetic composition further comprising a pharmaceutically acceptable carrier or cosmetically acceptable carrier.

9. The composition of claim 1, wherein the composition further comprises one or more agents xenogenic to the activated blastemas.

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

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

12. A method of preparing a composition comprising activated amphibian blastemas, wherein the method comprises injuring the amphibian, allowing the amphibian to recover from the injury, isolating blastemas from an injury site of the amphibian less than 7 days after theinjury, and activating the isolated blastemas to induce release of intracellular components from the blastemas, thereby preparing a composition comprising activated amphibian blastemas.

13. The method of claim 12, wherein activating the isolated blastemas comprises mechanical, chemical, and / or electrical disruption causing cell lysis to induce release of intracellular components from the blastemas.

14. The method of claim 12, wherein activating the isolated blastemas comprises mechanical disruption comprising using a blender, a homogenizer, a sonicator, or a freeze-thawing process, or manually grinding the isolated blastemas and optionally wherein the homogenizer is an ultrasonic homogenizer.

15. The method of claim 12, where activating the isolated blastemas comprises chemical disruption comprising using anionic, non-ionic, or zwitterionic detergent to lyse the cells.

16. The method of claim 12, wherein activating the isolated blastemas comprises using one or more detergents comprising sodium dodecyl sulfate (SDS, anionic), Triton X-100 (non- ionic, Triton X-114 (non-ionic), NP-40 (non-ionic), Tween 20 (non-ionic), Tween 80 (non- ionic), CHAPS (zwitterionic) and CHAPSO (zwitterionic).

17. The method of claim 12, wherein activating the isolated blastemas comprises electrical disruption comprising irreversible electroporation.

18. The method of claim 12, wherein activating the isolated blastemas comprises cytolysis or osmotic lysing.

19. The method of claim 12, wherein activating the isolated blastema comprises lysing the cells in saline solution, and optionally wherein the saline is phosphate buffered solution or physiological saline solution.

20. The method of claim 12, wherein the amphibian is a frog, toad, newt, or salamander.

21. The method of claim 12, wherein the amphibian is a young amphibian, and optionally, wherein the young amphibian comprises a froglet, tadpole, Urodele, or larval stage young Apoda.

22. The method of claim 12, wherein the amphibian is a neotenic Urodele.

23. The method of claim 12, wherein the method comprises isolating blastemas from the injury site between 12 hours to 7 days after injury, from 1 to 6 days, 1 to 5 days, 1 to 4 days, 2 to 4 days, or 3 days after injury 24. The method of claim 12, wherein injuring the amphibian comprises any method that induces blastema formation.

25. The method of claim 12, wherein injuring the amphibian comprises amputating a limb or tail or causing a wound and optionally wherein the wound is a skin wound.

26. The method of claim 12, wherein the method further comprises preparing the activated blastema into a gel, a paste, a solution, a serum, an extract, a cream, a lotion, a dispersion, a powder, or an emulsion.

27. A method of treating a wound or treating or preventing a skin disease or condition in a subject comprising administering the composition of the activated amphibian blastema of any one of claims 1-11 or the activated amphibian blastema obtained by the method of any one of claims 12-26 to the subject.

28. The method of claim 27, wherein the skin condition comprises fine lines and / or wrinkles, aging, redness, abrasion, burns, blisters, cuts, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, increasing skin elasticity and / or firmness, improving skin hydration, inflammation, or hyperpigmentation.

29. The method of claim 27, wherein preventing a skin condition comprises protecting the skin from damages caused by UV rays and / or environmental pollution.

30. The method of claim 27, wherein the method further comprises treating the skin with a laser before administering the activated blastema, and optionally wherein the laser is ablative fractional laser.

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