Colored biological wound treatment that provides monitoring of healing progress

Abstract

Provided are tissue regeneration wound treatments, methods of producing the tissue regeneration wound treatments, and methods of treating wounds using the tissue regeneration wound treatments. The tissue regeneration wound treatments include a skin substitute and a colorant applied to the skin substitute. The colorant is a biocompatible colorant that degrades upon attack by proteases within the treated wound.

Description

【Technical Field】 【0001】 The present disclosure generally relates to wound treatments and methods for stabilizing, protecting, and / or healing damaged tissue, and particularly to wound treatments and methods that indicate whether in-growth of the wound treatment into the skin substitute has occurred. 【Background Art】 【0002】 Healthy skin performs several different functions, including protecting underlying tissues from abrasions, microorganisms, water loss, and damage from ultraviolet light. The nervous system of healthy and normal skin also provides the sense of touch of contact, pressure, and vibration, the thermal sensations of heat and cold, and the sensation of pain. The body's thermoregulation depends on the ability of the skin to sweat and control blood flow to the skin to increase or decrease heat loss. Healthy skin contains three different tissue layers: a thin outer layer of cells called the epidermis, a thick middle layer of connective tissue called the dermis, and an inner subcutaneous layer. The thin outer layer of the epidermis is composed of flat, keratinized, dead keratinocytes that form a barrier against water loss and microbial invasion. The dead keratinocytes are derived from living keratinocytes in the basal layer above the dermis and are responsible for the re-epithelialization of the skin. The epidermis contains no nerves or blood vessels and takes up water and nutrients via diffusion from the dermis. The dermis beneath the epidermis consists mostly of collagen fibers produced by fibroblasts and some elastic fibers, which together with water and large proteoglycan molecules form the extracellular matrix (ECM). This skin layer provides mechanical strength and a substrate for the diffusion of water and nutrients. It contains blood vessels, nerves, sweat glands, hair follicles, and cells involved in immune function, growth, and repair. The subcutaneous layer is composed of adipocytes that form a thick layer of adipose tissue. 【0003】 A wound can be considered a disruption of the structural and functional integrity of the skin. Thus, a "wound" can include an injury that causes a cut, laceration, and / or disruption of the skin, such as, for example, a tear, abrasion, incision, puncture, detachment, burn, or other similar injury. 【0004】 After hemostasis, which is often performed following a wound event, the wound goes through the major healing stages of inflammation, proliferation, and remodeling. Chronic wounds can be considered wounds that have not gone through the normal healing process in an orderly and timely manner. Chronic wounds often remain in the inflammatory stage. 【0005】 In many cases, skin substitutes are frequently used to aid in the wound healing process and to more quickly restore at least some of the functions of healthy skin described above, especially in cases of serious wounds such as wounds that spread over a wide area or deep wounds, large or severe burns, or chronic wounds. Skin substitutes can be broadly considered as a group of elements or materials that enable temporary or permanent closure of wounds. Skin substitutes can generally be classified into biological skin substitutes, synthetic skin substitutes, or hybrid skin substitutes that include both biological and synthetic skin substitutes. 【0006】 Biological skin substitutes often have a more intact extracellular matrix structure, while synthetic skin substitutes can be synthesized on demand and tailored to specific purposes. Both biological and synthetic skin substitutes have their own advantages and disadvantages. Biological skin substitutes allow for the construction of a more natural new dermis and, due to the presence of a basement membrane, can enable superior re-epithelialization properties. Synthetic skin substitutes may be chemically synthesized, offering the advantage of increased control over scaffold composition. Synthetic skin substitutes include, for example, synthetic collagen or protein-based matrices, or synthetic biolayers containing collagen or protein-based components combined with silicone components. Hybrid skin substitutes can be partially synthesized or produced by living cells and partially chemically synthesized. 【0007】 Regardless of whether a biological, synthetic, or hybrid skin substitute is used, the purpose of using a skin substitute is to provide effective, timely, and scar-free wound healing while restoring as much of the pre-wound skin function as possible. 【0008】 Examples of commercially available synthetic skin substitutes include Biobrane®, Dermagraft®, Integra®, Apligraf®, MatriDerm®, OrCel®, Hyalomatrix®, and Renoskin®. 【0009】 U.S. Patent Application Publication No. 2003 / 0059460 discloses a hybrid polymer skin substitute material comprising synthetic and natural polymers that can be used for the regeneration of biological tissue. The hybrid includes cross-linked naturally occurring polymers and biodegradable, absorbable synthetic polymers. However, producing the hybrid material requires a series of complex process steps. In addition, the resulting hybrid material contains naturally occurring materials in addition to synthetic materials. 【0010】 Modern wound care products are so-called wet-to-dry wound dressings that promote improved wound healing by maintaining an appropriate moisture level on the wound. These products typically collect wound exudate and are replaced regularly. 【0011】 Biological skin substitutes may include, but are not limited to, autologous skin grafts, syngeneic skin grafts, allogeneic skin grafts, xenogeneic skin grafts such as porcine skin grafts, cadaveric allogeneic skin grafts, and skin grafts containing amniotic membrane tissue. 【0012】 Furthermore, in recent years, a new type of biological skin graft product has emerged that is intended to improve the wound microenvironment by providing shelter for proliferating cells. Typically, these new products are made from biological materials containing intact or reconstructed collagen. Examples include brands such as Oasis, Matristem, Integra, and Puracol. These products are often referred to as matrix products by clinicians. Matrix products are inserted into the wound, where they induce inward growth of cells. A secondary wet-to-dry wound dressing is then applied over the wound dressing. An example of a matrix product derived from intact decellularized fish skin is described in U.S. Patent No. 8,613,957, granted on December 24, 2013. The decellularized fish skin product described in U.S. Patent No. 8,613,957 functions as a scaffolding material that provides an intact scaffold for supporting the inward growth of endothelial and / or epithelial cells. Decellularized fish skin scaffolding materials are biocompatible and can be incorporated by the host. Omega3 Wound is a commercially available skin substitute made from minimally processed skin of wild-caught Atlantic cod native to Iceland. Structurally, fish skin is similar to human skin, having three basic layers including the epidermis, dermis, and subcutaneous tissue, and contains proteins, lipids, fatty acids, and other bioactive compounds homologous to human skin. 【0013】 Other examples of biological skin substitutes include the one described in U.S. Patent No. 6,541,023, which describes the use of a porous collagen gel derived from fish skin for use as a tissue engineering scaffold. The preparation of the collagen gel involves grinding the fish skin. In addition, Chinese Patent No. 1068703 describes a process for preparing fish skin for bandaging burns, which includes separating the fish skin from the fish body and placing the skin in a preservation solution of iodine tincture, ethanol, borneol, zinc sulfadiazine, and hydrochloric acid in sufficient quantities to establish a pH value of 2.5-3. However, these products can be difficult to handle, as the product in U.S. Patent No. 6,541,023 is in gel form and the product in Chinese Patent No. 1068703 is stored in solution. 【0014】 In addition, numerous extracellular matrix products for medical use are derived from human skin (ALLODERM® Regenerative Tissue Matrix (LifeCell)), fetal bovine dermis (PRIMATRIX® Dermal Repair Scaffold (TEI Biosciences)), porcine bladder (MATRISTEM® Extracellular Matrix Wound Sheet (Medline Industries, Inc.)), and porcine small intestine submucosa (OASIS® Wound Matrix (Healthpoint Ltd.)). 【0015】 As mentioned above, during healing, wounds go through three main stages: inflammation, proliferation, and remodeling. In the inflammatory stage, the body secretes proteases into the wound to remove damaged tissue and debris. In some cases, if skin substitutes such as extracellular matrix are inserted into the wound, proteases attack the skin substitutes and break them down as if they were damaged tissue or debris. In other cases, skin substitutes such as extracellular matrix function as intended, with inward cell growth and providing shelter for newly proliferating cells. 【0016】 Clinicians using matrix products typically examine the wound 1 to 3 days after the initial application of the matrix product to the wound bed. A significant challenge identified by the inventors is that clinicians and healthcare professionals , inferior Modified skin substitute It consists of necrotic tissue and pus, and skin substitutes such as matrix. It is moist and penetrated by inward growth of the cell. thing The inability to easily and / or accurately distinguish between them. The inventors of this invention, Deteriorated Skin substitutes and proper healing Skin substitute in wounds That is, for example 、 In the case of matrix skin substitutes, penetration occurs through intrinsic growth of cells. skin We have found that distinguishing between skin substitutes and other materials is crucial for efficient wound healing. If the added skin substitute material degrades, or if any part of it degrades, the degraded skin substitute material must be removed, the wound cleansed, and the sloughed-off tissue and pus, which are often present in the degraded skin substitute material, must be removed. After cleansing and removing the sloughed-off tissue and pus, a new treatment of matrix material can be applied to the wound. However, if it is determined that the added matrix material has penetrated as intended by inward cell growth, the matrix is ​​left in place, and monitoring of the matrix material continues until the wound heals properly. 【0017】 For example, when using fish skin-derived cell scaffold products to heal wounds (as described in U.S. Patent No. 8,613,957, granted on December 24, 2013), the inventors have found that clinicians and caregivers unconsciously misjudge or otherwise struggle to distinguish the wound healing scaffold from infection. This may be at least in part due to the color and / or odor associated with the wound healing scaffold as it decomposes and begins to integrate with the surrounding tissue. This may sometimes have a color similar to infected tissue (e.g., purulent infection) and may be mildly aromatic, leading some to interpret it as an odor similar to infected tissue. 【0018】 Therefore, the inventors have further identified that without an efficient and effective means of determining whether the skin substitute is penetrating through intrinsic cell growth, unnecessary removal or replacement of bandages is required to inspect the wound, wound exposure, and unnecessary reapplication of the fabricated skin substitute, which would hinder proper wound healing. 【0019】 In addition, infection is a major challenge in wound healing and management. For example, in the case of combat wounds, the morbidity and mortality rates of wounded soldiers on the battlefield are determined by infection. Infection accounts for one-third of all casualties, prolongs treatment, and increases the risk of amputation. Due to the unique mechanisms of injury and the harsh environment, combat wounds are more susceptible to contamination and more difficult to treat. An early sign of infection is an imbalance of bacteria within the wound. Common pathogens found in early-stage wounds include both Gram-positive (G+) and Gram-negative (G-) strains. Once an infection develops, the emergence of Gram-negative bacteria and multidrug-resistant (MDR) bacteria is observed. There is a strong need for effective and immediate intervention to reduce the risk of infection, benefit soldiers, and act as a force-building tool in the combat zone. 【0020】 Therefore, in addition to providing a means for determining whether a skin substitute is being penetrated by intrinsic cell growth, the inventors have further identified the challenge of ensuring that the skin substitute itself reduces or makes infection less likely. [Overview of the project] 【0021】 To address the above-mentioned problems, the present inventors hereby disclose an ingrowth-indicatory wound treatment comprising a skin substitute and a colorant added to the skin substitute. The colorant is a biocompatible colorant that degrades upon protease attack within the treated wound. 【0022】 Furthermore, a wound treatment method is provided, which includes providing a tissue regeneration wound treatment composition, applying the tissue regeneration wound treatment composition to a wound bed, and determining whether a skin substitute is being decomposed by protease attack within the wound by determining a change in the color of a colorant. The tissue regeneration wound treatment includes a skin substitute and a colorant added to the skin substitute. The colorant is a biocompatible colorant that decomposes upon being attacked by protease within the treated wound. 【0023】 A method for producing a tissue regeneration wound treatment is provided, which includes providing a skin substitute and adding a colorant to the skin substitute. The colorant is a biocompatible colorant that decomposes upon being attacked by protease within the treated wound. 【0024】 According to the embodiments described herein, the skin substitute is a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute. 【0025】 According to one or more embodiments, the skin substitute is an autologous skin graft, an isologous skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft. 【0026】 According to one or more embodiments, the skin substitute includes a scaffold material. 【0027】 According to one or more embodiments, the skin substitute includes a scaffold material including an extracellular matrix product. 【0028】 According to one or more embodiments, the extracellular matrix product is in the form of particles, sheets, or meshes. 【0029】 According to one or more embodiments, the skin substitute is a scaffold material including intact decellularized fish skin, and the intact decellularized fish skin includes an extracellular matrix material. 【0030】 According to one or more embodiments, the colorant includes a thiazine dye, a triarylmethane dye, or a combination of a thiazine dye and a triarylmethane dye. 【0031】 According to one or more embodiments, the coloring agent includes methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV). 【0032】 According to one or more embodiments, the skin substitute is freeze-dried, wherein a colorant is added to the skin substitute before freeze-drying or refrozen freezing of the skin substitute. 【0033】 According to one or more embodiments, the coloring agent is added to the skin substitute by staining it with a dye solution containing 0.01% to 0.0001% by weight of the coloring agent in deionized water or phosphate-buffered saline. 【0034】 According to one or more embodiments, the coloring agent is characterized by having one or more properties among those of an antibiotic, preservative, antibacterial agent, antiviral agent, antifungal agent, antiparasitic agent, anti-inflammatory agent, or antioxidant agent. 【0035】 According to one or more embodiments, the tissue regeneration wound treatment further includes a further active agent comprising one or more of the following: antibiotics, antiseptics, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, anti-inflammatory agents, antioxidants, drugs, proteins, peptides, or combinations thereof. 【0036】 According to one or more embodiments, the coloring agent does not cause permanent discoloration of the wound during healing. 【0037】 As described herein, the tissue regeneration treatments and methods disclosed herein provide accurate, efficient, and effective means for monitoring the progress of wound healing and for distinguishing between degraded applied skin substitutes and skin substitutes that have successfully penetrated through intrinsic cell growth applied to a wound. This makes it possible for clinicians or healthcare professionals to easily distinguish whether (1) the wound needs to be cleaned and the failed skin substitute, accompanied by sloughed-off tissue and pus, needs to be removed, or (2) the applied skin substitute should be left in place, wherein a colorant is added to the skin substitute and the colorant is degraded by protease attack within the treated wound. That is, the colorant is added to the skin substitute, for example, during the manufacturing stage, and the color of the colorant is altered, removed, or degraded by one or more proteases within the treated wound. 【0038】 This summary is provided to introduce, in a simplified form, a selection of concepts that will be further explained in the detailed description below. This summary is not intended to identify the main or essential features of the claimed subject matter, nor is it intended to be used as a scope of the claimed subject matter. 【0039】 Additional features and benefits of this disclosure are set forth in the following description, some of which may become apparent from that description or may be known through the practice of this disclosure. These features and benefits of this disclosure may be realized and obtained by the instructions and combinations specifically indicated in the appended claims. These and other features of this disclosure may become more apparent from the following description and the appended claims or may be known through the practice of the disclosure as set forth below. 【0040】 These and other features, aspects, and advantages of the present invention as disclosed herein will be best understood by referring to the following description, the appended claims, and the appended drawings. [Brief explanation of the drawing] 【0041】 [Figure 1A] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 1B] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 1C] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 1D] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 1E] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 1F] Examples of skin substrate embodiments according to this disclosure are provided below. [Figure 2A] Embodiments of skin substrates according to this disclosure in the form of decellularized fish skin are illustrated. [Figure 2B] Embodiments of skin substrates according to this disclosure in the form of decellularized fish skin are illustrated. [Figure 2C] Embodiments of skin substrates according to this disclosure in the form of decellularized fish skin are illustrated. [Figure 2D] Embodiments of skin substrates according to this disclosure in the form of decellularized fish skin are illustrated. [Figure 2E] Embodiments of skin substrates according to this disclosure in the form of decellularized fish skin are illustrated. [Figure 3] Examples of colored skin substitutes according to embodiments of this disclosure are provided. [Figure 4A] Examples of various colored skin substitutes according to embodiments of this disclosure are provided. [Figure 4B] Examples of various colored skin substitutes according to embodiments of this disclosure are provided. [Figure 4C] Examples of various colored skin substitutes according to embodiments of this disclosure are provided. [Figure 5] Examples of colored skin substitutes according to embodiments of this disclosure are provided. [Figure 6A] Examples of various mordanted and colored skin substitutes according to embodiments of this disclosure are provided. [Figure 6B] Examples of various mordanted and colored skin substitutes according to embodiments of this disclosure are provided. [Figure 6C] Examples of various mordanted and colored skin substitutes according to embodiments of this disclosure are provided. [Figure 6D] Examples of various mordanted and colored skin substitutes according to embodiments of this disclosure are provided. [Figure 7] Examples of various colored skin substitutes stained under pH grading according to embodiments of this disclosure are provided. [Figure 8] Examples of colored skin substitutes according to embodiments of this disclosure are provided. [Figure 9A] Examples of colored skin substitutes according to embodiments of this disclosure before and after exposure to collagenase are provided. [Figure 9B] Examples of colored skin substitutes according to embodiments of this disclosure before and after exposure to collagenase are provided. [Figure 10A] The images show the patient's wound before and after treatment using the methods and embodiments of this disclosure. [Figure 10B] The images show the patient's wound before and after treatment using the methods and embodiments of this disclosure. [Figure 11A] The images show the patient's wound before and after treatment using the methods and embodiments of this disclosure. [Figure 11B] The images show the patient's wound before and after treatment using the methods and embodiments of this disclosure. [Figure 12A] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12B] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12C] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12D] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12E] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12F] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12G] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12H]This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12I] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12J] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12K] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12L] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 12M] This disclosure illustrates the methods and embodiments of the treatment of a patient's wound before, during, and after the treatment. [Figure 13] This disclosure describes exemplary methods for treating wounds using tissue regeneration wound treatment according to embodiments of this disclosure. [Figure 14A] The results of bacterial inhibition / reduction assays according to embodiments of this disclosure are illustrated. [Figure 14B] The results of bacterial inhibition / reduction assays according to embodiments of this disclosure are illustrated. [Figure 14C] The results of bacterial inhibition / reduction assays according to embodiments of this disclosure are illustrated. [Figure 15A] A comparison of skin grafts according to embodiments of this disclosure is illustrated. [Figure 15B] A comparison of skin grafts according to embodiments of this disclosure is illustrated. [Figure 15C] A comparison of skin grafts according to embodiments of this disclosure is illustrated. [Figure 16] This shows an embodiment of a crosslinked and dyed scaffolding material. [Figure 17] Another embodiment of crosslinked and dyed scaffolding material is shown. [Figure 18] Another embodiment of crosslinked and dyed scaffolding material is shown. [Figure 19A] This shows a comparison of the colorfastness of crosslinked and dyed scaffolding materials in various embodiments. [Figure 19B]This shows a comparison of the colorfastness of crosslinked and dyed scaffolding materials in various embodiments. [Figure 20A] This shows a comparison of the colorfastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20B] This shows a comparison of the colorfastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20C] This shows a comparison of the colorfastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20D] This shows a comparison of the colorfastness of other embodiments of crosslinked and dyed scaffolding materials. 【0042】 The drawings are not necessarily drawn to scale. Instead, they are drawn to provide a better understanding of the components and are intended to provide illustrative explanations, not to limit the scope. The drawings illustrate the wound treatment and its features and exemplary configurations of its subcomponents as described herein. [Modes for carrying out the invention] 【0043】 A better understanding of the various embodiments of this disclosure can be obtained from the following description, along with the accompanying drawings, where similar reference numerals refer to similar elements. 【0044】 While various modifications and alternative structures are possible, specific exemplary embodiments are shown in the drawings described below. However, this disclosure is not intended to limit itself to the specific embodiments disclosed. On the contrary, it should be understood that the intent of the invention is to encompass all modifications, alternative structures, combinations, and equivalents that fall within the spirit and scope of the invention. 【0045】 The references used are provided for convenience only and do not define the scope of protection or the embodiments. 【0046】 Unless a term is expressly defined in this application to have the meaning described herein, it is understood that there is no intention to explicitly or indirectly limit the meaning of such term beyond its simple or ordinary meaning. 【0047】 Any element in a claim that does not explicitly state “means” or “processes” for carrying out a specific function should not be construed as a “means” or “process” section as defined in 35 U.S.C § 112. 【0048】 (1 or more) skin substitutes As described above, many different types of skin substitutes can be used to aid the wound healing process and to more quickly restore at least some of the functions of healthy skin. Skin substitutes can be broadly considered as a group of elements or materials that enable temporary or permanent closure of wounds. Skin substitutes can generally be classified into biological skin substitutes, synthetic skin substitutes, or hybrid skin substitutes that include both biological and synthetic skin substitutes. 【0049】 Examples of such skin substitutes are shown in Figures 1A to 1F. 【0050】 Figure 1A shows an example of a wound treatment skin substitute 100 according to one embodiment, comprising a first shredded decellularized fish skin particle 102. Figure 1B shows an example of a wound treatment skin substitute 110 according to one embodiment, comprising a second shredded decellularized fish skin particle 112. Figure 1C shows an example of a wound treatment skin substitute 120 according to one embodiment, comprising a third shredded decellularized fish skin particle 122. In the embodiments of Figures 1A, 1B, and 1C, the decellularized fish skin scaffold material is biocompatible and can be incorporated by the host. An example of such a commercially available decellularized fish skin scaffold material is Omega3® Wound by Kerecis, which is made from minimally processed skin of wild-caught Atlantic cod, as described in U.S. Patent No. 8,613,957. 【0051】 Other examples of applicable skin substitutes are shown in Figures 1D, 1E, and 1F. Figure 1D shows an example of a skin substitute 130 produced from processed tilapia fish skin. Tilapia-based skin grafts may be available in various sizes, including large skin grafts 132, medium skin grafts 134, and small skin grafts 136. Figure 1E shows an example of porcine skin grafts 140, including non-mesh porcine skin grafts 142 and mesh porcine skin grafts 144. Further examples of skin substitutes are shown in Figure 1F, in which case a synthetic skin substitute 150 is a bioengineered skin substitute formed from a bilayer tissue 152. In this non-limiting example, the bilayer tissue 152 of the dermal layer of the synthetic skin substitute 150 is a type I bovine collagen gel seeded with living human neonatal fibroblasts. The epidermis is neonatal keratinocytes. Cells actively secrete growth factors, cytokines, and extracellular matrix (ECM) proteins. A non-limiting example of such synthetic skin substitutes is Apligraf®, which can be used to treat diabetic foot ulcers and venous leg ulcers. 【0052】 Referring here to Figures 2A and 2B, exemplary embodiments of decellularized fish skin pieces 200, 210 are described. An exemplary cross-section of decellularized fish skin 200, prepared as described in U.S. Patent No. 8,613,957, is illustrated in Figure 2A in size given by the size of a user's gloved hand 202. The size of the decellularized fish skin 200 is not limited and may be produced or provided or trimmed to fit the size and shape of the wound being treated. Furthermore, although the decellularized fish skin shown is non-mesh fish skin, mesh decellularized fish skin may also be used. 【0053】 It should be understood that decellularized fish skin can be granulated, ground, or otherwise processed into various sizes and shapes. As shown in Figure 2B, multiple decellularized fish skin sheets 210 can be similar in size and shape (e.g., rectangular) to the decellularized fish skin sheet 200 in Figure 2A, or they can have more uniform dimensions (e.g., square) shapes, such as the decellularized fish skin sheet 220 exemplified in Figure 2B. 【0054】 The decellularized fish skin scaffolds 210, 220 shown in Figures 2A and 2B are substantially rigid and inelastic in their freeze-dried form. The decellularized fish skin scaffolds can be treated with one or more enzymes that act to increase their ductility and / or elasticity. In some embodiments, the enzymes act by cleaving interconnected extracellular matrix components without substantially affecting the healthy properties that are important for wound preservation and / or stabilization. In some embodiments, the enzymes cleave covalent bonds within and / or between elastin, proteoglycans, collagen, or other extracellular matrix materials, but the modified decellularized fish skin retains a substantial portion of the extracellular matrix contents, even if partially removed from its natural three-dimensional structure. 【0055】 In some embodiments, enzymatic treatment negatively affects the use of modified decellularized fish skin as a scaffolding material. However, it should be understood that, surprisingly, the loss of function as a scaffolding material does not significantly affect the use of decellularized fish skin as a wound preservation and stabilization material. Thus, the ductility and / or elasticity of the material can be increased while maintaining the composition of extracellular components, and despite this potentially negatively impacting the use of the material as a scaffold for wound healing, modified decellularized fish skin can still function as a wound preservation / stabilization material. 【0056】 Decellularized fish skin scaffolds can be ground and provided in the form of particles. It should be understood that the size of individual ground particles may vary depending on the type and / or method of grinding. For example, decellularized fish skin particles can be produced via a jet milling process designed to output particles smaller than a specified size. In some embodiments, the decellularized fish skin is cut, shredded, or ground into particles, which may be done in a measured manner to produce uniform particles or may be done roughly, thereby producing particles of various different sizes. 【0057】 Figure 2C illustrates an exemplary depiction of large particles 232 of granulated or pulverized decellularized fish skin 230 obtained by crushing a sheet of decellularized fish skin scaffolding material with a grinder, for example, a cannabis grinder. Figure 2D illustrates an exemplary depiction of filamentous cotton-like fibers 242 of granulated or pulverized decellularized fish skin 240 obtained by crushing a sheet of decellularized fish skin scaffolding material with a grinder according to embodiments of the present disclosure. Figure 2E is an exemplary depiction of small, powdery particles 252 of pulverized decellularized fish skin 250 obtained by crushing a sheet of decellularized fish skin scaffolding material with a grinder, for example, a cannabis grinder. 【0058】 In embodiments, the wound treatment is or comprises at least one particle-formed, in particular, shredded decellularized fish skin particle of a predetermined size. The particle-formed, i.e., shredded decellularized fish skin particle is configured to provide a scaffold material for supporting cell migration, adhesion, proliferation, and differentiation to promote tissue repair and / or replacement, as described in U.S. Patent No. 8,613,957, filed on October 6, 2010, and granted on December 24, 2013. 【0059】 The extracellular matrix (ECM) of vertebrates is a complex structural entity that surrounds and supports cells. The ECM is composed of a complex mixture of structural proteins, most abundantly collagen, along with other specialized proteins and proteoglycans. The scaffolding materials described herein are nearly intact cell-free scaffolds of natural biological ECM components from fish skin. The scaffolds may also contain lipids naturally occurring from fish skin. The native three-dimensional structure, composition, and function of the dermal ECM remain essentially unchanged, providing a scaffold that supports cell migration, adhesion, proliferation, and differentiation, and thus facilitating tissue repair and / or replacement. 【0060】 The scaffolding material according to the present invention is obtained from intact fish skin. Any type of fish, including bony and cartilaginous fish, can be used as a source of fish skin. For example, the source may be round fish such as cod, haddock, and catfish; flatfish such as halibut, flounder, and sole; salmonids such as salmon and trout; mackerel such as tuna; or small fish such as herring, anchovies, mackerel, and sardines. In certain embodiments, the fish skin is obtained from fatty coldwater fish and / or fish known to contain large amounts of omega-3 oil. Examples of fish rich in omega-3 oil include salmon, sardines, tuna, herring, cod, sardines, mackerel, sablefish, smelt, white fish, hoki, and several types of trout. 【0061】 Fish skin is removed from the fish before processing. If the fish skin is from a species of fish that has scales, the scales must be removed from the fish skin so that most of the scales are removed, or at least the hydroxyapatite is removed from the scales. The expressions "most of the scales removed" or "substantially scale-free" mean that at least 95%, preferably at least 99%, and more preferably 100% of the scales on the fish skin are removed. "Substantially scale-free" fish skin may also refer to fish skin from a species of fish that does not have scales. Scales are removed before all processing using purely mechanical pressure (e.g., via a knife, abrasive shaking, water pressure, a special scale removal device using the same mechanical force as a knife, or other pressure devices such as polishing with ceramic or plastic), or after any chemical processing (e.g., decellularization) using mechanical pressure to wash away the scales. If the fish skin is initially treated chemically and / or enzymatically (e.g., with TRITON® X-100), the mechanical pressure should generally be gentle because it is susceptible to tearing after decellularization. Scales can be removed in more than one step; for example, they can be partially removed before decellularization, followed by further removal during and / or after decellularization. Alternatively, scales can also be removed by chemical treatment alone. 【0062】 After the scales are removed, the fish skin is optionally frozen before decellularization. To preserve the collagen structure of the scaffold, the fish skin can be rapidly frozen by incubating the skin in liquid nitrogen or by using other specialized freezing equipment capable of freezing the skin to below -70°C. Alternatively, the fish skin can also be frozen in conventional freezers typically found in seafood processing plants. The freezing process can help thaw or partially thaw the cells containing the intact fish skin, thus facilitating decellularization. Once frozen, the fish skin can be thawed for further processing later. 【0063】 Fish skin can be washed with a buffer before further processing, regardless of whether it has been frozen. For example, fish skin can be disinfected and stabilized by washing it 1 to 3 times with a buffer optionally containing one or more antioxidants (e.g., ascorbic acid (e.g., 50 mM ascorbic acid), vitamins A, C, E, and beta-carotene), antibiotics (e.g., streptomycin and penicillin), proteases (e.g., dispase II), and protease inhibitors (e.g., antipain, aprotinin, benzamidine, bestatin, DFP, EDTA, EGTA, leupeptin, pepstatin, phosphoramidone, and PMSF). The buffer may have a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. The buffer can also be used as a culture medium to preserve the fish skin for several days to several weeks or more. In certain embodiments, the fish skin is stored in the buffer at a temperature of approximately 4°C. 【0064】 After freezing and / or washing and / or storage in buffer, the fish skin is treated with one or more decellularizing solutions to remove antigenic cellular material from the fish skin with minimal to no damage to the mechanical and structural integrity and biological activity of the naturally occurring extracellular matrix. 【0065】 As used herein, the term “extracellular matrix” or “ECM” refers to non-cellular tissue material present within fish skin that provides structural support to skin cells, in addition to performing a variety of other important functions. The ECM described herein does not necessarily include matrix material that is entirely composed of or reformed from extracted, purified, or isolated ECM components (e.g., collagen). However, in some embodiments, the ECM used as a skin substitute may include matrix material that is entirely composed of or reformed from extracted, purified, or isolated ECM components (e.g., collagen). 【0066】 As used herein, terms such as “cell-free,” “decellularized,” and “decellularized fish skin” refer to fish skin from which a substantial amount of cellular and nucleic acid contents have been removed, leaving the complex three-dimensional interstitial structure of the extracellular membrane (ECM). In embodiments, “decellularized fish skin” may further include fish skin containing omega-3 polyunsaturated fatty acids (PUFAs) in addition to the complex three-dimensional interstitial structure of the ECM that is free from a substantial amount of cellular and nucleic acid contents. 【0067】 A “decellularizing agent” is an effective agent for removing a substantial amount of cellular and nucleic acid contents from the extracellular membrane (ECM). The ECM is considered “decellularized” or “substantially devoid” of cellular and nucleic acid contents (i.e., “substantial” amounts removed) when at least 50% of viable and unviable nucleic acids and other cellular material are removed from the ECM. In certain embodiments, approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of viable and unviable nucleic acids and cellular material are removed. Decellularization can be confirmed, for example, by examining the DNA content of the treated fish skin. Nucleic acid removal from ECM can be determined, for example, by histological examination of the ECM and / or by biochemical assays such as the PICOGREEN® assay, diphenylamine assay, or PCR. 【0068】 Decellularization involves disrupting the cell membrane and releasing the cellular contents. Decellularization may involve one or more physical treatments, one or more chemical treatments, one or more enzymatic treatments, or any combination thereof. Examples of physical treatments include sonication, mechanical agitation, mechanical massage, mechanical pressure, and freezing / thawing. Examples of chemical decellularizers include ionic salts (e.g., sodium azide), bases, acids, detergents (e.g., nonionic and ionic detergents), oxidizing agents (e.g., hydrogen peroxide and peracids), hypotonic solutions, hypertonic solutions, chelating agents (e.g., EDTA and EGTA), organic solvents (e.g., tri(n-butyl) phosphate), ascorbic acid, methionine, cysteine, maleic acid, and polymers that bind to DNA (e.g., poly-L-lysine, polyethylimine (PEI), and polyaminedoamine (PAMAM)). Nonionic detergents include 4-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol, t-octylphenoxypolyethoxyethanol, polyethylene glycol tert-octylphenyl ether (TRITON® X-100) (Dow Chemical Co.). Ionic detergents include sodium dodecyl sulfate (SDS), sodium deoxycholate, TRITON® X-200, and amphoteric detergents (e.g., CHAPS). Other suitable decellularizing detergents include polyoxyethylene (20) sorbitan monooleate and polyoxyethylene (80) sorbitan monooleate (Tween 20 and 80), 3-[(3-chloramidopropyl)-dimethylamino]-1-propane sulfonate, octyl glucoside, and sodium dodecyl sulfate. Examples of enzymatic decellularizing agents include proteases, endonucleases, and exonucleases. Proteases include serine proteases (e.g., trypsin), threonine proteases, cysteine ​​proteases, aspartate proteases, metalloproteases (e.g., thermolysin), and glutamate proteases. Decellularization is generally performed at a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and above 10.0.In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. 【0069】 An example of a decellularization process is to incubate fish skin in a solution containing 1 M NaCl, 2% deoxycholic acid, 0.02% sodium azide, and 500 ppm streptomycin. In another example, fish skin is incubated in a first decellularization solution containing a protease (e.g., 2.5 U / mL Dispase II) and other components (e.g., 0.02% sodium azide). The first decellularization solution is drained, and the fish skin is then treated with a second decellularization solution, such as a solution containing a detergent (e.g., 0.5% TRITON® X-100) and other components (e.g., 0.02% sodium azide). In another example, fish skin is first treated with a decellularizing solution containing a detergent (e.g., 0.5% TRITON® X-100) along with other components (e.g., 0.02% EDTA, sodium azide, and / or deoxyforic acid), and then incubated in a second decellularizing solution containing a detergent such as SDS. 【0070】 The fish skin may or may not be cultured with shaking. The decellularization steps (one or more) can be repeated as needed by pouring out the remaining decellularization solution, optionally washing the fish skin with a buffer (e.g., Hanks equilibrium salt solution), and then re-treating the fish skin with another decellularization step. Once a sufficient amount of cellular material has been removed, the decellularization solution can be removed (e.g., by aspiration or by gently pouring out the solution). 【0071】 After decellularization, the fish skin can be optionally washed with water, buffer, and / or salt solutions. Examples of suitable washing solutions include Dulbecco's phosphate-buffered saline (DPBS), Hanks' equilibrium salt solution (HBSS), Medium 199 (M199, SAFC Biosciences, Inc.), and / or L-glutamine. The washing steps (one or more) are generally performed at a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. 【0072】 To improve the appearance of the final product, the fish skin may be bleached as desired. Bleaching can be performed before, after, and / or concurrently with decellularization. For example, one or more bleaching agents can be incorporated into one or more and / or one or more buffers of the decellularization solution. Examples of bleaching agents include sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate. In certain embodiments, when strong bleaching agents such as (one or more) persulfates are used, bleaching and decellularization can be combined in a single step involving incubation of the fish skin in a mixture of one or more bleaching agents, a thickener, and a peroxide source. For example, a dry bleaching mixture can be prepared (see, for example, “Bleaching Mixture” described in Example 5), and then water, hydrogen peroxide, or a combination thereof can be added to the dry mixture to form a bleaching solution that may also be sufficient for decellularization. The bleaching agents (e.g., sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate) should be in a dry mixture at approximately 40-60% w / w. The combination of EDTA and persulfates can be added to the mixture to accelerate decellularization in addition to bleaching. 【0073】 In certain embodiments, the concentration of EDTA in the dry mixture is about 0.25–5% w / w. Hydrogen peroxide may be about 15–25% of the mixture, and the peroxide sources may be sodium percarbonate and potassium percarbonate. Sodium phosphate perhydrate and sodium carbonate or magnesium metasilicate and silicic acid can also be used as peroxide sources. The dry mixture may also contain, for example, 1–10% w / w of silica and hydrated silica, and optionally one or more stearates (e.g., ammonium stearate, sodium stearate, and / or magnesium stearate). In addition, the dry mixture may optionally contain thickeners such as hydroxypropyl methylcellulose, hydroxyethylcellulose, algin (i.e., alginates), organic gums (e.g., cellulose, xanthan gum), sodium metasilicate, and combinations thereof, to increase the viscosity of the bleaching / decellularizing solution and protect protein fibers from damage. Bleaching, and / or bleaching + decellularization, is generally carried out at a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. After bleaching and / or bleaching + decellularization, the fish skin is optionally washed with a solution containing L-glutamine under the pH conditions described above. 【0074】 In certain embodiments, the fish skin is treated with digestive enzymes. Similar to bleaching, digestion can be performed before, after, and / or concurrently with decellularization. Suitable enzymes include proteases, such as serine proteases, threonine proteases, cysteine ​​proteases, aspartate proteases, metalloproteases, and glutamate proteases. In certain embodiments, the digestive enzyme is a serine protease such as trypsin. The digestive enzyme may be one that functions in an alkaline environment, limits cross-linking within the ECM, and softens the fish skin. Digestion is generally performed at a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and above 10.0. In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. 【0075】 Decellularized fish skin can optionally be cryopreserved. Cryopreservation may involve immersing the fish skin in a cryoprotectant solution before freezing. The cryoprotectant solution generally contains a suitable buffer, one or more cryoprotectants, and optionally a solvent, such as an organic solvent combined with water to minimize expansion and contraction. Examples of cryoprotectants include sucrose, raffinose, dextran, trehalose, dimethylacetamide, methyl sulfoxide, ethylene glycol, glycerol, propylene glycol, 2-methyl-2,4-pantandial, certain antifreeze proteins and peptides, and combinations thereof. Alternatively, if the decellularized fish skin is rapidly frozen (flash-frozen) before sublimation to minimize ice crystal formation during the freezing process, the fish skin can optionally be frozen in a buffer without cryoprotectants. Freezing is generally carried out at a pH of at least 5.5, such as 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example, between 7.5 and 8.5. 【0076】 Decellularized fish skin can be packaged in sterile containers such as glass vials or pouches. In one embodiment, TYVEK® pouches are used. For example, the fish skin can be incubated in a cryoprotectant solution, packaged in a TYVEK® pouch, and then frozen in a freeze-dryer at a rate compatible with the cryoprotectant. 【0077】 Decellularized fish skin can be freeze-dried, i.e., frozen, under low temperature and vacuum conditions so that water is sequentially removed from each ice crystal phase without the ice recrystallizing. During freeze-drying, water is generally removed first by sublimation and then, if necessary, by desorption. Another method for removing excess water after processing and before sterilization is vacuum press molding. 【0078】 In certain embodiments, decellularized fish skin is sterilized before and / or after freezing. Sterilization methods are well known in the art. For example, decellularized fish skin can be placed in an ethylene oxide chamber and treated with an appropriate cycle of ethylene oxide. Other sterilization methods include sterilization with ozone, carbon dioxide, gaseous formaldehyde, or radiation (e.g., gamma rays, X-rays, electron beam treatment, and subatomic particles). 【0079】 Decellularized fish skin can be stored in a non-aqueous solution such as alcohol, as an alternative to, or in addition to, freezing, freeze-drying, and / or vacuum press molding. 【0080】 The resulting product (scaffold material) is a sterile, collagen-based matrix with properties that can promote tissue regeneration, repair, and / or replacement (e.g., repair, regeneration, and / or proliferation of endogenous tissue). The term “scaffold material” in relation to fish skin refers to materials including decellularized fish skin, which may be bleached, digested, freeze-dried, etc., as discussed above. Scaffold materials can provide an intact scaffold for supporting endothelial and / or epithelial cells, can be incorporated by a host, are biocompatible, do not significantly calcify, and can be stored and transported at ambient temperatures. The expression “incorporated by a host” means, as used herein, that the cells and tissues of a patient being treated with the scaffold material can proliferate within the scaffold material, and that the scaffold material is actually incorporated into / absorbed by the patient’s body. The term “biocompatible” refers to a material that is substantially non-toxic in the in vivo environment of its intended use and is not substantially rejected by the patient’s physiological system (i.e., non-antigenic). 【0081】 This can be measured by the ability of a material to pass biocompatibility tests specified in the International Organization for Standardization (ISO) standard number 10993, titled "Use of International Standard ISO-10993, Biological Evaluation of Medical Devices Part 1: Evaluation and Testing," and / or United States Pharmacopeia (USP) 23 and / or U.S. Food and Drug Administration (FDA) Blue Book Memorandum number G95-1. Typically, these tests measure the toxicity, infectivity, pyrogenicity, irritation, reactivity, hemolytic activity, carcinogenicity, and / or immunogenicity of the material. A biocompatible structure or material will not cause a seriously harmful, long-lasting, or aggravated biological reaction or response when introduced into the majority of patients, and is distinct from the mild, transient inflammation typically associated with the surgery or implantation of foreign bodies into living organisms. 【0082】 The scaffold material contains proteins from the extracellular matrix (ECM) of fish skin. ECM components within the scaffold material can include, for example, structural proteins, adhesive glycoproteins, proteoglycans, non-proteoglycan polysaccharides, and matrix cell proteins. Examples of structural proteins include collagen (the most abundant protein in the ECM), such as fibrous collagen (types I, II, III, V, and XI), fatty collagen (types IX, XII, and XIV), short-chain collagen (types VIII and X), basement membrane collagen (type IV), and other collagens (types VI, VII, and XIII), as well as elastin and laminin. Examples of adhesive glycoproteins include fibronectin, tenascin, and thrombospondin. Examples of proteoglycans include heparin sulfate, chondroitin sulfate, and keratan sulfate. An example of a non-proteoglycan polysaccharide is hyaluronic acid. Matrix cell proteins are a structurally diverse group of extracellular proteins that regulate cellular functions through interactions with cell surface receptors, cytokines, growth factors, proteases, and the extracellular matrix (ECM). Examples include thrombospondin (TSP) 1 and 2, tenascin, and SPARC (an acidic, cysteine-rich secretory protein). 【0083】 In certain embodiments, decellularization (and other optional processing steps) does not remove all naturally occurring lipids from the lipid layer of fish skin. Therefore, the scaffold material may contain one or more lipids from the fish skin, particularly from the lipid layer. For example, the scaffold material may contain up to approximately 25% w / w of lipids (of the dry weight of the entire scaffold material after freeze-drying), such as 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 24% w / w of lipids. The presence of lipids in the scaffold material can be confirmed, for example, by organic solvent extraction followed by chromatography. Examples of suitable organic solvents include acetone and chloroform. 【0084】 Lipids in the scaffolding material may include, for example, fatty acid acyls (i.e., fatty acids, their complexes, and derivatives), glycerolipids, glycerophospholipids (i.e., phospholipids), sphingolipids, glycolipids, polyketides, sterollipids (i.e., sterols), certain fat-soluble vitamins, prenolipids, and / or polyketides. Examples of fatty acid acyls include saturated fatty acids such as polyunsaturated fatty acids, fatty acid esters, fatty amides, and eicosanoids. In certain embodiments, fatty acids include omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are found in high concentrations in fish oil. Other fatty acids found in fish oil include arachidic acid, gadoleic acid, arachidonic acid, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, behenic acid, erucic acid, and lignoceric acid. Examples of glycerolipids include mono-, di-, and tri-substituted glycerols such as monoacylglycerols, diacylglycerols, and triacylglycerols (i.e., monoglycerides, diglycerides, and triglycerides). Examples of glycerophospholipids include phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Examples of sphingolipids include phosphorus sphingolipids and sphingoglycolipids. Examples of sterollipids include cholesterol, steroids, and secosteroids (various forms of vitamin D). Examples of prenolic lipids include isoprenoids, carotenoids, and quinones such as vitamins E and K, as well as hydroquinones. 【0085】 The scaffolding material may contain one or more additional active agents (i.e., agents added during or after processing the scaffolding material), such as antibiotics, preservatives, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, and anti-inflammatory agents. The active ingredient may be a compound or composition that promotes wound care and / or tissue healing, such as an antioxidant or agent. It may also be a protein or proteins and / or other biological substance. Antibiotics, preservatives, and antibacterial agents may be added in amounts sufficient to provide the scaffolding material with effective antibacterial properties. In certain embodiments, the antibacterial agent is one or more antibacterial metals, such as silver, gold, platinum, copper, zinc, or a combination thereof. For example, silver may be added to the scaffolding material during processing in the form of an ion, metal, element, and / or colloid. Silver may be combined with other antibacterial agents. Anti-inflammatory agents may be added in amounts sufficient to reduce and / or suppress inflammation in the area of ​​the wound or tissue to which the scaffolding material is applied. 【0086】 Scaffolding materials can be used in a dry form. Alternatively, scaffolding materials can be rehydrated before use. In certain embodiments, one or more scaffolding materials are laminated together to form a thicker scaffolding material. 【0087】 Generally, scaffolding materials are approximately 0.1 to 4.0 mm thick (i.e., thickness in cross-section), such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, or 3.5 mm. The thickness can depend on numerous factors, including the type of fish used as the starting material, processing, freeze-drying, and / or rehydration. Of course, if the product contains more than one layer of scaffolding material, the thickness will increase proportionally. 【0088】 The shredded decellularized fish skin particles of the wound treatment and method embodiments provide, as an advantage, a sterile, collagen-based matrix that has properties that can promote the regeneration, repair, and / or proliferation of tissues such as endogenous tissue, while being configured to form or be added to the wound to conform well to the shape of the wound. In embodiments, the shredded decellularized fish skin particles are configured to be packed into wounds, such as eroded or tunnel-shaped wounds, in a manner that is not possible with sheet-based materials. That is, the shredded decellularized fish skin particles may be configured to promote incorporation, i.e., the patient's cells and tissues being treated with the scaffold material may be configured to proliferate within the scaffold material, and the scaffold material may be configured to be actually incorporated / absorbed into the patient's body. 【0089】 The shredded decellularized fish skin particles according to the embodiments may be configured to actively promote wound healing as a physical scaffold for infiltration into cells involved in wound healing / repair, such as intrinsic cell growth and angiogenesis. The shredded decellularized fish skin particles of the wound treatment embodiments may, as an advantage, be configured to retain the three-dimensional ("3D") structure of the decellularized fish skin, which has an extracellular matrix ("ECM") that is recognizable with respect to histological analysis, for example. The dimensions of the shredded decellularized fish skin particles may be further configured to facilitate shaping, packaging, or otherwise applying the shredded decellularized fish skin particles into the wound cavity with greater precision than existing approaches to wound treatment. 【0090】 In the embodiment, the shredded decellularized fish skin particles have a maximum dimension within a range of a maximum value of a predetermined size threshold and a minimum value of a size threshold that is effective in preserving the matrix structure of the decellularized fish skin and promoting the regenerative inward growth of cells into the wound. That is, the maximum dimension, such as the maximum dimension of the length, width, and / or thickness of the shredded decellularized fish skin particles, may be smaller than a maximum size such as 1 mm and larger than a minimum size such as the size at which the ECM is destroyed. In the embodiment, the shredded decellularized fish skin particles are obtained by providing a decellularized fish skin sheet as described above, then shredding the decellularized fish skin sheet, and optionally sieving the shredded particles until the shredded decellularized fish skin particles are within a predetermined minimum and maximum size threshold. 【0091】 The shredded, decellularized fish skin particles can be further configured to resist shear forces, taking their dimensions into consideration. Thus, the shredded, decellularized fish skin particles can provide improved wound care for patients who are moving or being moved between locations or environments, or during normal patient activities such as walking during recovery. 【0092】 The shredded, decellularized fish skin particles of the embodiment can, as an advantage, be applied topically to wounds and / or implanted to provide a scaffold for intrinsic cell growth and angiogenesis, and can further provide additional benefits including tissue scaffold formation benefits such as adhesion barriers, soft tissue repair, and dehiscence prevention. 【0093】 (one or more) colorants Various examples of colorants are conceivable. In its broadest sense, a colorant, or a combination of colorants, provides color to a skin substitute that changes or disappears in color based on changes in the state of the wound or changes in the skin substitute during the healing process. In a preferred embodiment, the colorant is degraded by attack by one or more proteases within the wound. With such a colorant, the colorant disappears upon degradation by one or more proteases. For example, a colorant may provide a blue or purple color to a skin substitute. However, upon attack by one or more proteases within the wound after application of a wound treatment containing the skin substitute and colorant, the color of the skin substitute of the wound treatment is also degraded or disappears, thereby changing the color of the applied wound treatment to the original color or another color of the skin substitute. However, the color change of a colorant is not limited to this and may also include color shifts associated with changes in the state of the wound. For example, the color provided by a colorant may be induced so that the original color of the skin substitute does not change upon application or addition of the colorant. However, changes in the color of the coloring agent can be induced or caused by changes in the condition of the wound, thereby changing the skin substitute of the wound treatment to a new color, or a color different from the original color of the skin substitute. 【0094】 Dyes may be used as colorants. Preferred examples of colorants are thiazine dyes such as methylene blue (MB). The structure of methylene blue (MB) is provided below: [ka] 【0095】 Methylene blue (MB), also known as methylthioninium chloride or Basic Blue 9, is a cationic thiazine dye used in a variety of applications, including fabric dyeing, medical treatment, and research. It is used to treat methemoglobinemia at doses up to 2 mg / kg over several hours. 【0096】 Another embodiment of the colorant is a triarylmethane dye. A preferred example of a triarylmethane dye is gentian violet (GV), which has the following structure: [ka] 【0097】 Gentian violet (GV), also known as crystal violet, methyl violet 10B, or hexamethyl para-losaniline chloride, is a triarylmethane dye commonly used for histological staining in Gram staining. Topical gentian violet (V) is used to treat certain types of oral fungal infections (thrush) and fungal infections of the skin. 【0098】 Another embodiment of the colorant is brilliant blue FCF (BB-FCF) having the following structure: [ka] 【0099】 Also known as Blue No. 1, Brilliant Blue FCF (BB-FCF) is a triarylmethane dye primarily used as a blue coloring agent for processed foods, pharmaceuticals, dietary supplements, and cosmetics. It is one of the oldest FDA-approved coloring additives and is generally considered non-toxic and safe. 【0100】 Another embodiment of the coloring agent is indigo carmine (IC) having the following structure: [ka] 【0101】 Indigo carmine (IC), also known as Food Blue 1, is an organic salt derived from indigo by aromatic sulfonation, which makes the compound water-soluble. It is blue at pH below 11.4 and yellow at pH above 13.0, and can also be used as a redox indicator, turning yellow when reduced. 【0102】 Other dye chemicals or dye mixtures, including the following, may be used and have been investigated by the inventors: 【0103】 Woad Powder (HUE-3023), a wool dye proposed by The Woolery, has an INCI (International Nomenclature for Cosmetic Ingredients) for its extract of *Talus tinctorius*. It is a commonly used dyeing chemical for yarns and garments, typically used for dyeing in alkaline environments. Woad Powder is considered a useful colorant due to its powdery properties, which allow it to bind to keratin proteins. 【0104】 The coloring additive, D&C Green #5 Powder AN0725, is made from natural sources and is generally used in cosmetics. The INCI name for this coloring additive is Green No. 5. The powder is a dry, powdery, water-based dye. It can be selected as a coloring agent due to its typical water-based cosmetic color in powder form. 【0105】 The coloring additive, Ultra Marine Blue H9-03R1, is used in cosmetics, including eye makeup (not for lip products), soaps, and lotions. This coloring additive is based on natural resources and has the INCI name Ultramarine Na6Al6Si6O24S4. It is an oil-dispersible pigment, insoluble in water and oil, and has CAS number 57455-37-5. It is considered highly effective in cosmetics due to its strong coloring power and its insolubility in water and oil when added to cosmetics, making it a viable choice as a colorant. However, this coloring additive may contain undesirable residues, which should be considered. 【0106】 FD&C Blue #1 is a liquid coloring additive used in cosmetics, soaps, bath salts, and bath bombs. It is made from natural resources and has the INCI name Blue No. 1. This coloring additive can be supplied as a pre-mixed water-based dye. It is a typical water-soluble liquid dye for cosmetic use. 【0107】 The coloring additive liquid, D&C Green #5, is also used in cosmetics, soaps, bath salts, and bath bombs. It is made from natural resources and has the INCI name Green No. 5. This coloring additive can be supplied as a pre-mixed water-based dye. It is a typical water-soluble liquid dye for cosmetic use. 【0108】 The coloring additive liquid, D&C Green #6 Oil AM4299, is also used in cosmetics, soaps, bath salts, and bath bombs. It is made from natural resources and has the INCI names Green No. 6 and caprylic / capric triglyceride. This coloring additive is supplied as a pre-mixed oily liquid dye, blended, for example, in fractionated coconut oil, to extend its shelf life. This dye may be considered a product that reacts better with oily dyes, and subsequently withstands washing processes well and does not dissolve in hydration. However, it should be noted that when using this dye, measures should be taken to mitigate or address the possibility of permanent staining of the wound, which could result in a tattoo-like effect on the patient. 【0109】 Green Concentrated Food Coloring is a food coloring manufactured by Rayner. It has the INCI names Water, Tartrazine (E102) (1.87%), Brilliant Blue FCF (E133) (0.13%), and Acetic Acid. Because it is supplied as a pre-mixed aqueous liquid food coloring mixture, it can be considered a coloring agent. The dye mixture is considered a harmless dye mixture. 【0110】 Garnie Natural Color, Mahogany Brown contains: Aqua, Deceth-3, Alles-12, Cocamidomypa, Oleth-30, Ammonium Hydroxide, Deceth-5, Glycerin, Oleic Acid, Oleyl Alcohol, Hexadimethrin Chloride 2, 4-Diaminophenoxyethanol HCl, p-Aminophenol, m-Aminophenol, Ascorbic Acid, Hydroxyethylcellulose, Sodium Metabisulfite, Ethanolamine, Wheat Germ Oil, Thioglycerin, Polyquaternium-6, Toluene-2, 5-Diamine, Polyquaternium-67, 2-Methyl-5-Hydroxyethylaminophenol, Ammonium Thiolactate, and Simmondsia Chinensis Oil. This is a hair colorant manufactured by Garnier, containing the ingredients of jojoba seed oil, isopropanolamine, resorcinol, EDTA, and parfum. This dye is commercially available as a pre-mixed hair color kit. This dye is formulated to bind to proteins and react with collagen in scaffolds or skin substitutes, making it another embodiment of a colorant. 【0111】 ELEA, Color & Care, Black is a hair colorant manufactured by ELEA. It contains the following ingredients: Aqua, Ceteryl Alcohol, Ammonia Ceteret-20, Cetrimonium Chloride, Cocamdopropyl Betatan, Oleic Acid, Propylene Glycol, PEG-40, Hydrogenated Histrol Oil, p-Phenylenediamine, 2,4-Diaminophenoxyethanol, HCl, Vitis Vinifera Seed Oil, Sodium Metabisulfite, Erythorbic Acid, Parfum, Coumarin, Limonene, Linalool, Resorcinol, and Tetrasodium EDTA. This dye is formulated to bind to proteins and react with collagen in scaffolds or skin substitutes, making it another embodiment of a colorant. 【0112】 Various examples and embodiments of colorants are provided herein, but this description of possible colorants is not, and should not be considered, exhaustive of all possible colorants, either as dyes or coloring additives. 【0113】 Addition of colorants to skin substitutes In preferred embodiments, methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV) may be used as colorants added to the skin substitute. 【0114】 In embodiments where methylene blue (MB) and gentian violet (GV) are used in combination, methylene blue (MB) and gentian violet (GV) are used together in equal weight ratios. However, in other embodiments, methylene blue (MB) and gentian violet (GV) are used together in different weight ratios. Other embodiments include any combination of these dyes, methylene blue (MB) and gentian violet (GV), and any two or more other dyes. Exemplary methods and embodiments are described below. 【0115】 In the first embodiment, a decellularized fish skin scaffolding material made from minimally processed skin of wild-caught Atlantic cod native to Iceland is provided as a skin substitute. For brevity, in the following subsections, unless otherwise specified, this scaffolding material made from minimally processed skin of wild-caught Atlantic cod native to Iceland will be referred to as the “scaffold” or “scaffolding material” provided as an embodiment of the skin substitute. 【0116】 The following describes a method for adding one or more colorants to a scaffold to improve the fastness of the colorants. 【0117】 In a preferred embodiment, a common process is used to add methylene blue (MB) and / or gentian violet (GV) colorants to the scaffold. 【0118】 In the exemplary procedure, 100 mL of dye solution was used, based on either deionized water or phosphate-buffered saline (hereinafter abbreviated as "PBS") containing 0.001 wt% of each colorant (MB and GV) (0.002 wt% in total, or 20 mg / L if two dyes (MB and GV) were used). Liquefied scaffold pieces measuring approximately 4 × 4 cm and weighing 0.25–0.30 g were added to the solution and left for 3 hours. Then, the Kroma scaffolds were removed from the solution, washed with tap water, rinsed with deionized water, and frozen. The resulting stained scaffold 300 is shown in Figure 3. 【0119】 As described above, when MB and GV are used, the total amount of both dyes in the scaffold is approximately 1 mg / g. The exact same method can be used with any combination of the four dyes mentioned above (methylene blue (MB), gentian violet (GV), brilliant blue FCF (BB-FCF), and indigo carmine (IC)), or any of these four dyes, per the same total concentration (0.002 wt%) or dye concentration (0.001 wt%) in water or PBS solution. Even slight changes in the total concentration or volume of the dye solution will yield the same total concentration in the scaffold for any single dye or any other combination of the dyes listed above. A UV-VIS spectrophotometer can be used (and has been used by the inventors) to measure the absorbance and some concentration of the colored solution before and after the staining process with any single dye or any combination of dyes to determine its affinity for adsorption to the scaffold. 【0120】 Figures 4A, 4B, and 4C show the scaffolding materials obtained by staining with 100 mL of dye solution based on either deionized water or phosphate-buffered saline (hereinafter abbreviated as "PBS"), with scaffolding material 410 in Figure 4A containing 0.001% by weight of MB, scaffolding material 420 in Figure 4B containing 0.001% by weight of GV, and scaffolding material 430 in Figure 4C containing a total amount of 0.001% by weight of MB / GV in a 25 / 75 ratio, after standing for 24 hours. 【0121】 Other embodiments have used other additional combinations of dyes, including: (1) BB-FCF and IC applied together using the same experimental setup as above (when MB and GV are applied); (2) BB-FCF and / or IC applied before and after MB and / or GV to extend lifetime under in vivo conditions; and (3) BB-FCF and / or IC combined with MB and / or GV. In the above combinations, the solvent could be either water or PBS. Other embodiments include other solvent mixtures which will be discussed in later sections herein. Figure 5 shows scaffold 510 obtained by staining with BB-FCF and IC applied in the same weight as in the above examples having colorants MB and GV as described above, with a total concentration of 0.002 wt% and 0.001 wt% of each colorant BB-FCF and IC, after standing for 3 hours. 【0122】 Alternative methods using mordants In other embodiments, various methods or color fasteners are used to improve the fastness of the colorants or combinations of colorants added to the scaffolding material. 【0123】 Mordants, or dye fasteners or fixatives, are a group of compounds used in biological dyeing and the textile industry, primarily composed of divalent metals (salts). Compounds such as tannic acid and tartaric acid cream (potassium salt of tartaric acid) are often used for the same purpose, but these are generally not considered true mordants. 【0124】 The choice of mordant often depends on the dye being used; for example, some zinc salts are used with MB, and iodine (KI+12) may be used with GV. Although iodine is also used as a mordant in Gram staining, it is considered a scavenger rather than an actual mordant. 【0125】 One definition of a mordant is a polyvalent metal ion that forms a coordination complex with a particular dye; however, this definition is not necessarily limited to the term “mordant” in this disclosure, and as reflected above, some compositions (e.g., tannic acid, tartrate cream, iodine) are considered mordants by those skilled in the art, even though they generally do not fit this definition. 【0126】 Mordants can generally be applied to the coloring process in three ways: (1) pre-mordanting (on-chrome), where the substrate is treated with the mordant and then with the dye; (2) meta-mordanting (metachrome), where the mordant is present in the coloring solution from the beginning (this process is simpler than pre-mordanting / post-mordanting but is only applicable to a limited number of dyes); and (3) post-mordanting (after-chrome), where the substrate is treated with the dye first and then with the mordant. 【0127】 Figures 6A and 6B show mordanted scaffolding materials; Figure 6A shows a mordanted sample of Kroma scaffolding material 610 dyed with a 0.002 wt% MB / GV combination using alum, and Figure 6B shows a pre-mordanted sample of scaffolding material 620 dyed with a 0.002 wt% MB / GV combination using alum. Alum, also known as aluminum sulfate, is one of the most commonly used mordants in textiles because it provides good colorfastness to a variety of dyes, as well as enhancing color brightness and saturation. However, it is by no means the only mordant that can be used with scaffolding. In other embodiments, possible mordants / salts include, but are not limited to, NaCl, MgCl2, MgSO4, CaCO3, CaCl2, KCl, ZnCl2, several other Zn salts, or KI / I2 may also be used. 【0128】 In other embodiments, as shown in Figures 6C and 6D, variations in metamordant are observed. In Figure 6C, scaffolding material 630 is stained with an MB / GV combination at a total concentration of 0.002% by weight, to which 0.5 grams of CaCl2 is added. In Figure 6D, scaffolding material 640 is stained with an MB / GV combination at a total concentration of 0.002% by weight, to which 0.5 grams of NaCl is added. 【0129】 In embodiments, any one or more combinations of these metal salts / mordants are used with any of the above dyes or any combination of dyes, or any other combination of dyes and coloring techniques. 【0130】 In different embodiments, all three mordanting methods are applied at approximately 90°C for approximately 2 hours. If this is not possible, the scaffold substrate can be kept in the solution at room temperature for more than 48 hours. In our case, our experimental results show that heating the scaffold in a sodium chloride solution at approximately 80°C for 2 hours can substantially decompose the collagen into a more gel-like form, which is likely due to the partial decomposition of collagen into gelatin. Therefore, a “cooling method,” or a hybrid method, for example, at approximately 37°C for 12 hours, is preferred. 【0131】 Meta-mordanting is generally considered the most restrictive method. This is due to various factors, including the solubility of the dye-mordant complex formed during the process (known as dye lake). The solubility of the complex is often lower than that of the mordant and dye, causing them to precipitate separately, thus limiting the combinations of mordants and dyes that can be applied. Furthermore, when using meta-mordanting, the time in the solution depends on the mordanting time, which is approximately two days at room temperature. Therefore, the dyeing process may need to be longer or carried out at higher temperatures. The amount of adsorbed color has been shown to be directly related to the time in the solution and possibly correlated with temperature as well. 【0132】 Preferred embodiments include pre-mordanting or post-mordanting, where post-mordanting is potentially more preferable for our application, given that the scaffold does not lose much color during the mordanting process, because post-mordanting can be used without changing the current coloring and quantification methods. The pre-mordanting process can alter the rate of dye adsorption because the dye lake forms directly on the surface of the scaffold during adsorption. Furthermore, the mordant can "leak" into the dye solution, potentially causing precipitation of dye molecules or changes in absorbance intensity. In either case, this can cause problems when measuring the amount of pigment adsorbed onto the scaffold. 【0133】 As mentioned above, alum is a preferred mordant, but in other embodiments, other mordants, including metal salts such as sodium, magnesium, potassium, and iron salts, may be used, though not limited to those mentioned above. 【0134】 pH gradient staining or "through staining" In other embodiments, a “through-staining” process was used for staining the scaffold. This process involves a stepwise change in the pH of the dye solution during the staining process, from a slightly basic pH 9–10 to a slightly acidic pH 3–4. This can be achieved using various weak acids / weak bases (e.g., acidic acid / sodium bicarbonate), diluted solutions of strong acids / strong bases (e.g., HCl / NaOH), or very small amounts of concentrated strong acids / strong bases, or a combination of these methods. This process can be used in place of or in conjunction with any of the staining methods described herein. The effect of through-staining is thought to be due to the limited stability of the tertiary structure of the protein (collagen in this case) across the pH scale. When exposed to a pH that the protein has not developed to handle, the protein structure deforms by opening, exposing the dye binding sites. 【0135】 Figure 7 shows various scaffold materials obtained by applying pH grading to the staining process. First, scaffold material 710 is shown after staining with the MB / GV combination at a total concentration of 0.002 wt% with a pH gradient applied. Next, scaffold material 720 is shown after staining with MB at a total concentration of 0.002 wt% with a pH gradient applied. Next, scaffold material 730 is shown after staining with the MB / BB-FCF combination at a total concentration of 0.002 wt% with a pH gradient applied. Finally, scaffold material 740 is shown after staining with BB-FCF at a total concentration of 0.002 wt% with a pH gradient applied. 【0136】 A staining or fixation method different from conventional methods. In other embodiments, different dyeing or color-fixing methods are used. The following is a description of some of the different dyeing methods used in other embodiments. In these embodiments, the effectiveness of color absorption or fixation was determined primarily by visual inspection. 【0137】 In some embodiments, alternative solvents were used in the dyeing process. The dyeing methods described above were carried out using aqueous / water-based solvents for the dyes. However, the four dyes mentioned above (MB, GV, BB-FCF, IC) are not only soluble in water but also in various solvents such as ethanol, and are slightly lipophilic. 【0138】 In some embodiments, MB and / or GV are dissolved in ethanol, and the lyophilized scaffold is stained in the ethanol solution. As a result, much brighter coloration was obtained, even when a more concentrated dye solution and a longer staining time were applied, compared to similar aqueous methods. While brighter colors were visually observed overall in these embodiments, these embodiments may be considered effective and preferable as they still provide an effectively colored skin substitute, and may tend to have improved color fastness and a lower likelihood of permanent tattooing of the wound. 【0139】 In other embodiments, the dye was dissolved in oleic acid, but in these embodiments, the solubility was lower. However, using a 70 / 30 oleic acid / ethanol mixture resulted in higher solubility. The resulting scaffold color was found to be darker overall than that of ethanol and oleic acid at the same time and dye concentration. 【0140】 In other embodiments, vegetable oils were also used. Fish oil / cod liver oil can also be used. The use of oil / organic solvent-based dye solutions can be carried out with any combination of dyes and may also be carried out on a scaffold pre-mordanted with various oils, fatty acids, their salts, and solvents. 【0141】 In other embodiments, a coating treatment was performed after dyeing. Of particular interest in these embodiments are oil and sugar-based coatings. 【0142】 In one embodiment, a mixture of triglycerides, monoglycerides, and free fatty acids derived from fish oil was used for coating by spraying a thin film onto a scaffold after staining. This sample acquired some resistance to in vitro decolorization and degradation experiments compared to a similar sample without coating. Furthermore, this could also be done using a suitable fatty acid alkyl ester. 【0143】 In other embodiments, sugar-based coatings are made using a variety of sugars, either monosaccharides such as ribose, fructose, or dextrose, or disaccharides such as sucrose or maltose. The selected sugar is dissolved in an aqueous solution, the scaffold is immersed in it, and then lyophilized again. Non-reducing sugars may be dissolved in the coloring solution. Sugars can enhance the stability of the collagen itself by introducing additional crosslinking. Furthermore, sugars contain a large number of -OH groups, which can promote further binding to pigment molecules by hydrogen bonding or dipole forces. In addition, nitrogen-containing sugars such as N-acetylglucosamine can form covalent bonds with the free amino / acid ends of collagen and certain pigments. 【0144】 While almost all possible methods and embodiments described herein can be used together, using multiple components from each category, such as two or more mordants, to enhance color vividness, fastness, etc., can also increase the complexity, potential side effects, and overall cost of manufacturing the dye scaffold. 【0145】 Therefore, preferred methods and embodiments for generating suitable prototypes are similar to the “basic” staining process. The most notable issue identified is the lifespan of the dye under in vivo conditions (e.g., mouse). Possible improvements include increasing the binding of dye molecules within the collagen matrix of the scaffold by using a mordant step, a pH gradient, or a combination of the two. Two preferred embodiments of the stained scaffold are shown for comparison, as shown in Figure 8. Scaffold 810 is stained using an MB / GV combination at a total concentration of 0.002 wt%, and scaffold 820 is stained using a combination of MB / GV and a pre-mordant dye at a total concentration of 0.002 wt%. 【0146】 Decomposition of scaffolds by collagenase catalysts When scaffolding is used as a biological bandage, the body breaks down the large scaffolding into a "pool" of micro-fragments, which aids in the reconstruction and growth of the affected area. In the validation experiment, a PBS solution of collagenase was selected to mimic this scaffolding breakdown process and its effect on (one or more) pigments in the scaffolding sample. In nature and in humans, the primary function of collagenase is to break down collagen to the peptide level, which occurs, for example, in damaged tissue within the skin, helping the body generate new, healthy tissue. 【0147】 For this purpose, a stock solution of 0.50 mg / mL collagenase in PBS was prepared. In the first experiment, the stock solution was diluted to 10 μg / mL or 100 μg / mL. A total of nine solutions (10 ml) were prepared using either PBS or "human plasma-like solution" as the majority of the solution, and then scaffold pieces were placed in the solutions and maintained at room temperature for an extended period. 【0148】 The inventors discovered that unstained scaffolds begin to degrade in collagenase PBS solution. In the case of plasma-like medium, even when the collagenase concentration was 10 times higher than in the plasma solution, the solution appeared to inhibit collagenase, as seen by the comparative level of scaffold degradation after approximately 3 days. During this time, scaffolds stained with a 0.002% MB and GV combination showed little change. 【0149】 To degrade the MB / GV stain, a fairly high concentration of collagenase was applied. For this purpose, the original 0.5 mg / mL PBS solution was used. A series of variations were tested after testing unstained scaffold pieces for comparison of time and degradation levels. It was found that complete degradation into fine particles took only about 24 hours. Turning to the stained samples, the first scaffold sample 910 stained in a 0.001 wt% MB / GV solution was tested, as shown in Figure 9A. With a 0.5 mg / mL collagenase solution, complete degradation of that sample took about 2 days. As seen in Figure 9B, although some of the color has bled into the solution, the majority of the dye is still bound to the small collagen particles 920. 【0150】 The following five "prototypes" described in the previous section were tested in the same manner. The prototypes tested were: 1) MB / GV in water at 0.002 wt%, 2) MB / GV in PBS at 0.002 wt%, 3) MB / BB FCF in PBS at 0.002 wt%, 4) IC in water at 0.001 wt%, and 5) scaffold material stained with MB / GV in water at 0.002 wt%, coated with a mixture of triglycerides, monoglycerides, and free fatty acids derived from fish oil. Samples were checked over the following four days, and in all cases except one (Sample 4: IC in water at 0.001 wt%), total degradation took four days. 【0151】 These experiments showed that the stained scaffolds (1-5 above) decomposed into fine fragments, and in all cases, a significant portion of the dye remained on those fragments. This suggests that the dye binds firmly to the collagen / peptides of the scaffold, not just to the surface. As mentioned above, in all cases except one (i.e., sample 4: 0.001 wt% IC in water), decomposition took approximately 4 days, and no significant differences were observed among the MB / GV stained samples. Samples 3 and 4 differed slightly; sample 4, stained with IC, decomposed almost completely in just under 24 hours, leaving only a few fragments. Sample 3 was more stable compared to sample 4, but decomposed faster than the other three samples, resulting in smaller fragments. 【0152】 In the embodiments described above, the coloring of the scaffold generally involved combining methylene blue (MB) and gentian violet (GV) in equal weight ratios. However, this is not always the case. The amount of colorant can be adjusted to produce a scaffold containing a specific amount of colorant, with the aim of keeping the amount of colorant below, and in some embodiments significantly below, the maximum permissible amount of MB issued by the FDA for this type of product. The amount of both dyes MB / GV combined in the scaffold is approximately 1 mg / g, but the maximum permissible amount may be 2 mg / g, 3 mg / g, 4, 5 mg / g, 6 mg / g, 7 mg / g, 8 mg / g, 9 mg / g, or even 10 mg / g in some embodiments. 【0153】 In some embodiments, the step of adding the colorants uses 100 mL of a dye solution based on (deionized water or PBS) containing 0.001% by weight of each colorant (0.002% by weight or 20 mg / L in total). However, this amount of colorant in the dye solution can be increased or decreased according to the needs of the skin substitute to which the colorant is applied. For example, the dye solution may contain 1.0 to 10.0% by weight of colorants or pigments (based on deionized water, PBS, or some other dye solvent), 1.0 to 20.0% by weight of colorants or pigments (based on deionized water, PBS, or some other dye solvent), 1.0 to 0.01% by weight of colorants or pigments (based on deionized water, PBS, or some other dye solvent), depending on the skin substitute to which the colorants or pigments are added. It may contain an amount of 0.01 to 0.001% by weight of a colorant or pigment (based on some other dye solvent), 0.05 to 0.002% by weight of a colorant (based on deionized water, PBS, or some other dye solvent), 0.01 to 0.0002% by weight of a colorant (based on deionized water, PBS, or some other dye solvent), or 0.01 to 0.0002% by weight of a colorant or pigment (based on deionized water, PBS, or some other dye solvent). 【0154】 A scaffold piece measuring approximately 4 x 4 cm and weighing 0.25-0.30 g may be added to the solution and left for 3 hours. However, as mentioned above, the size of the scaffold material and the time it is left in the staining solution can be changed. Furthermore, the size of the scaffold material can naturally be changed, and the volume and concentration of the dye solution may be adjusted as needed. The scaffold is then removed from the solution, washed with tap water, rinsed with deionized water, frozen, and freeze-dried. 【0155】 In some embodiments, when PBS is used instead of deionized water as the base of the solution, the relative amounts of MB and GV adsorbed by the scaffold change, from approximately 60% GV and 40% MB by weight relative to the aqueous solution to approximately 40% GV and 60% MB by weight relative to PBS. However, the total amount of adsorbed colorant remains almost the same. Furthermore, when MB and GV are used in combination, various MB / GV ratios may be used, including MB ratios of 95 / 5, 90 / 10, 85 / 15, 80 / 20, 75 / 25, 70 / 30, 65 / 35, 60 / 40, 55 / 45, 50 / 50, 45 / 55, 40 / 60, 35 / 65, 30 / 70, 25 / 75, 20 / 80, 15 / 85, 10 / 90, and 5 / 95. The MB / GV ratio can be in the range of 10-50% MB, 10-60% MB, 10-70% MB, 10-80% MB, and 10-90% MB, with the remaining corresponding percentages (90-50%, 90-40%, 90-30%, 90-20%, and 90-10%). In a preferred embodiment, the MB / GV ratio is 50 / 50. In another preferred embodiment, the MB / GV ratio is 75 / 25. And in yet another preferred embodiment, the MB / GV ratio is 25 / 75. 【0156】 In addition to the use of MB and GV as colorants for skin substitutes, other embodiments use food colorings, or more precisely, active compounds (dyes / pigments) in food colorings, in combination with or in place of MB and GV. 【0157】 In one embodiment, a lipid-soluble food coloring and one water-soluble food coloring were used. The dye in the lipid-soluble food coloring was E133, i.e., brilliant blue FCF (BB-FCF), which is a water-soluble molecule with a molecular structure very similar to GV. The dye made up about 40% by weight of the food coloring, with the remainder of the additives being for the "lipid-soluble" version. The dye in the water-soluble food coloring was E132, i.e., indigo carmine (IC), which made up about 85% by weight of the food coloring. 【0158】 The inventors have discovered that food coloring can be removed from scaffolding material by both enzymatic decomposition using collagenase and subsequent standing in a sodium bicarbonate solution of approximately 1 M. 【0159】 The binding mechanism of the aforementioned colorants, which has been found to bind to the collagen / peptides in the scaffolding material, allows for binding not only to the surface but also to other collagen or peptide-based skin substitutes via a similar binding mechanism using a similar or appropriate colorant. 【0160】 The scaffolding material according to the present invention can be obtained from intact fish skin or any species of fish, including bony or cartilaginous fish, which can be used as a source of fish skin. For example, the source may be round fish such as cod, haddock, and catfish; flatfish such as halibut, flounder, and sole; salmonids such as salmon and trout; mackerel such as tuna; or small fish such as herring, anchovies, mackerel, and sardines. Furthermore, other collagen, peptides, or other protein-containing skin substitutes, whether biological, synthetic, or hybrid skin substitutes, can be similarly colored by appropriate combinations of dyes, pigments, and / or other colorants. 【0161】 Tests and results in mice and patients mouse Embodiments of decellularized fish skin scaffolding material made from minimally processed skin of wild-caught Atlantic cod native to Iceland are provided as a skin substitute. Again, in the following subsections, unless otherwise specified, “fish skin” used as scaffolding material, made from minimally processed skin of wild-caught Atlantic cod native to Iceland, will be referred to as “scaffolding” or “fish skin” as “scaffolding material” provided as an embodiment of a skin substitute. 【0162】 A total of 52 mice were tested using embodiments of colored scaffolding material as a skin substitute. 【0163】 In the first pilot study, Pilot 1, four mice were treated. 【0164】 Pilot 1 included the following: 1) 0.005% by weight of MB + 0.005% by weight of GV colorant, fresh decellularized fish skin stained in water for 3 hours before freeze-drying; and 2) Fresh decellularized fish skin stained in water for 3 hours before freeze-drying with 0.010 wt% MB + 0.010 wt% GV colorants. 【0165】 In the second pilot study, Pilot 2, 16 mice were treated. 【0166】 Pilot 2 included the following: 1) Fresh decellularized fish skin stained in water for 24 hours before immersion in a cold sugar solution containing 0.001% by weight of MB + 0.001% by weight of GV coloring agent, and then freeze-dried; 2) Freeze-dried fish skin stained in water for 3 hours before immersion in a cold sugar solution containing 0.001% by weight of MB + 0.001% by weight of GV coloring agent; 3) 0.001% by weight of MB + 0.001% by weight of GV colorant, freeze-dried fish skin dyed in water for 3 hours before immersion in mineral oil and freeze-drying; and 4) Freeze-dried fish skin stained with 0.001% by weight of MB + 0.001% by weight of GV colorant, in water for 3 hours before freeze-drying. 【0167】 In the second and third pilot studies, 32 mice were treated. 【0168】 Pilot 3 included the following: Freeze-dried fish skin stained with 0.001% by weight of MB + 0.001% by weight of GV colorant in PBS for 3 hours before freeze-drying. 【0169】 The results of the respective mouse studies in Pilot 1, Pilot 2, and Pilot 3 yielded the following findings: No unexpected inflammation or other adverse events were detected after using colored fish skin as a scaffolding material. The treatment product (scaffolding material) decomposed within a normal time, and wounds healed normally. Importantly, no permanent or semi-permanent tattooing of the wound bed was detected. 【0170】 Human patient Three patients (Patient 1, Patient 2, and Patient 3) were treated with minimally processed skin from wild-caught Atlantic cod native to Iceland, which in this subsection is referred to as "fish skin," "scaffolding," or "scaffolding material." 【0171】 In each of the three patients (Patient 1, Patient 2, and Patient 3), scaffold materials were prepared in the same manner as in Pilot 3 above, using 0.001% by weight of MB + 0.001% by weight of GV colorant and freeze-dried fish skin that had been stained in PBS solution for 3 hours before freeze-drying. 【0172】 Patient 1 was treated with colored fish skin using the first photograph as shown in Figure 10A on October 12, 2021, and the same wound on Patient 1 was photographed again seven days later on October 19, 2021, as shown in Figure 10B. 【0173】 Patient 2 was treated with colored fish skin using the first photograph as shown in Figure 11A on October 25, 2021, and the same wound on Patient 2 was photographed again 7 days later on November 2, 2021, as shown in Figure 11B. 【0174】 Finally, various wounds in patient 3 were treated with colored fish skin from January 20, 2022 to February 10, 2022. Figures 12A–12N show the treated wounds each time the wound dressing was changed. Figure 12A shows the colored fish skin applied on day 0, and Figure 12B shows the same wound on day 4. Newly stained fish skin is applied as shown on day 6 in Figure 12C, and Figure 12D shows the treated wound on day 8, two days later. In the same patient 3, newly stained fish skin is applied to a different wound in Figure 12E, Figure 12F shows the same wound on day 2, and Figure 12G shows the same wound on day 5. Figure 12H shows the application of newly colored fish skin, Figure 12I shows the results on day 2, and Figure 12J shows the results on day 4. Finally, Figure 12K shows the application of the newly colored fish skin, Figure 12L shows the healing after 2 days, and Figure 12M shows the healing result after 4 days. 【0175】 In each of the patients 1-3 described above, no device-related inflammation or other adverse events were reported after the use of the colored fish skin. The applied treatment appears to promote the healing of these chronic wounds. Furthermore, the applied colored fish skin normally decomposed within the wound. In addition, no permanent or semi-permanent tattooing of the wound bed was detected after day 5. 【0176】 Further examples (For example, when healing wounds using Kerecis® fish skin-derived cell scaffold products as disclosed in U.S. Patent No. 8,613,957), as described above, the inventors have found a significant problem in that clinicians may unknowingly misidentify or otherwise struggle to distinguish wound healing scaffolds from infection. This may be at least in part due to the color and / or odor associated with wound healing scaffolds as they decompose and begin to integrate with the surrounding tissue, which may sometimes have a color similar to infected tissue (e.g., purulent infection) and may be mildly aromatic, leading some to interpret it as an odor similar to infected tissue. Thus, the inventors have found that there is a problem in the art in which there can be great benefit from improved products or improvements to known products. 【0177】 One solution is to pseudo-color the fish skin-derived cell scaffold so that it can be more easily identified in the clinic and / or distinguished from surrounding tissue when placed in a wound bed. To this end, the following disclosure provides exemplary data from a series of tests focused on identifying colorants that can remain stable over time and can be incorporated into the fish skin scaffold during processing / manufacturing. 【0178】 A first set of experiments was conducted to determine the stability of various colorants in the decellularization solution (referred to herein as “Decell solution”) used in the processing / manufacturing of Kerecis® fish skin-derived cell scaffold products, which are made from minimally processed skin of wild-caught Atlantic cod, as described in U.S. Patent No. 8,613,957. The Decell solution was prepared according to EBL M222, and the stability of six different colorants listed in the table below was tested. 【0179】 [Table 1] 【0180】 Decell solutions were prepared according to EBL M222. Each colorant was prepared to a solution strength of 1% w / v (for example, as listed in Table 1). 50 mL of Decell solution was divided equally into seven separate plastic tubes, each with a lid. The first tube contained only the Decell solution and served as a control. 0.5 mL aliquots of each of the six prepared color solutions were separately added to the corresponding tubes containing 50 mL of Decell solution. Any reactions or visible changes in the mixtures were monitored over time. 【0181】 The solutions in each tube containing each colorant were monitored 30 minutes and 24 hours after incubation and documented photographically at the start of the experiment. 【0182】 Many of the colorants were found to be quite bright in the Decell solution at the start. Within the first 20 minutes, most of the colorants began to fade, with a notable exception being methylene blue. This trend continued, and after 24 hours, all colored Decell solutions, except for the one with methylene blue added, had turned white or nearly white. Therefore, methylene blue is considered a preferred embodiment of a colorant to be added during the decellularization step in the manufacture of Kerecis® fish skin-derived cell scaffold products, made from minimally processed skin of wild-caught Atlantic cod, as described in U.S. Patent No. 8,613,957. 【0183】 In another embodiment, a method 1300 for treating a wound using a tissue regenerating wound treatment is provided, as shown in Figure 13. Step 1310 provides a tissue regenerating wound treatment comprising a skin substitute and a colorant, the colorant being a biocompatible colorant that degrades upon protease attack within the treated wound. Step 1320 applies the tissue regenerating wound treatment to the wound bed. Step 1330 determines whether the skin substitute is being degraded by protease attack within the wound by determining a change in the color of the colorant. 【0184】 In a further exemplary method, a tissue regenerating wound treatment, including a skin substitute, is in the form of a blue-colored (e.g., MG / GV) extracellular matrix that is inserted into the wound bed and a secondary wound dressing is applied over it. In yet another exemplary method, the color of the wound bed is recorded during wound examination. If the color is blue, the tissue regenerating wound treatment is considered (correctly) intact, and it is concluded that intrinsic cell growth is (correctly or probably) occurring. If the wound treatment is no longer blue, it has fallen off and needs to be washed away and a new material applied to the wound bed. 【0185】 The coloring materials used must be biocompatible and break down when proteases attack the matrix itself. This does not necessarily have to be permanent, although sometimes a permanent color or "tattoo effect" may remain on the wound after healing has occurred. 【0186】 Additional tests The first color test was performed on molted fish skin. Tests were then conducted on fish skin-based wound products to determine how the material responded to various dyes and chemicals. The objective was to determine how the fibrous collagen material reacted with various dyes and whether the reaction differed in wet and dry conditions. 【0187】 Test scheme Glass bowls, tweezers, and airtight plastic containers were used in the experiments. These tests were intended to answer questions such as how collagen materials react with various types of dyes, whether oil-based or water-based dyes react better with proteins, whether they are retained after washing, and at what point in the manufacturing process the dyeing of molted fish skin scaffolds is optimal. The various dyes / colorants / pigments / color additives tested included Wood Powder (HUE-3023); Color additive D&C Green #5 Powder AN0725; Color Additive Ultra Marine Blue H9-03R1; Color additive Liquid FD&C Blue #1; Color additive Liquid D&C Green #5; Color additive Liquid D&C Green #6 Oil AM4299; Green Concentrate Food Coloring; and Garnier Natural Color, Mahogany Brown. 【0188】 Coloring before freeze-drying In the first step, the decellularized fish skin is colored before freeze-drying. This is done to see how the material reacts with color when wet, and how the colorants react during washing and freeze-drying. The tests were performed after the decellularization process in the manufacture of wound products using decellularized fish skin. 【0189】 Decellularized fish skin scaffolds were kept in a dye chemical for 60 minutes, and then washed in continuous running water for 2 hours. 【0190】 Coloring after freeze-drying In the second step of this test, the material was colored after freeze-drying. This was to check whether there was a difference in the reaction between the scaffold and the color after freeze-drying, and whether the structure was more open to the dye chemicals. The sheet was then freeze-dried again. 【0191】 Test Procedure A single piece of skin was removed from the decellularized fish skin and cut into small pieces. Each piece was placed in a coloring solution, which consisted of either an undiluted solution, a mixture of coloring powder and water / oil, or a mixture of colorant and hair color developer. The pieces were left for two hours, then thoroughly washed, examined, and photographed. Promising pieces were immersed in water in a sealed container and stirred until the next morning. This was to see if the color would eventually become insoluble in water. All pieces were examined and washed again. All solutions were immersed in distilled water and colored after five minutes. Promising pieces were sent for freeze-drying (freezing at -80°C) and freeze-dried in a freeze dryer. 【0192】 A better understanding of the various embodiments of this disclosure can be obtained from the following description, along with the accompanying drawings, where similar reference numerals refer to similar elements. 【0193】 While various modifications and alternative structures are possible, specific exemplary embodiments are shown in the drawings described below. However, this disclosure is not intended to limit itself to the specific embodiments disclosed. On the contrary, it should be understood that the intent of the invention is to encompass all modifications, alternative structures, combinations, and equivalents that fall within the spirit and scope of the invention. 【0194】 The references used are provided for convenience only and do not define the scope of protection or the embodiments. 【0195】 Unless a term is expressly defined in this application to have the meaning described herein, it is understood that there is no intention to explicitly or indirectly limit the meaning of such term beyond its simple or ordinary meaning. 【0196】 As used herein, the term “treatment” is intended to be understood by its common dictionary definition. That is, the term “treatment” in a broad sense includes medical care and / or pharmaceuticals given to a patient for illness or injury. As will be understood by those skilled in the art, “treatment” includes the use of chemical, physical, or biological agents to preserve or impart certain properties to something. Thus, “treatment” can refer to medical care provided (i.e., a prescribed form of method or set of acts) or pharmaceuticals used to preserve or impart certain properties to something. 【0197】 As a non-limiting example, the particulate form of decellularized fish skin disclosed herein may be referred to as a “treatment,” i.e., a pharmacopoeia used to preserve and / or stabilize a wound, or to bring any of the other disclosed beneficial effects to a wound site. Similarly, in some examples, a treatment includes the use of the disclosed decellularized fish skin in particulate form within a method for stabilizing and / or protecting a wound. 【0198】 As used herein, terms such as “decellularization,” “decellularized fish skin,” and “cell-free fish skin” refer to fish skin prepared by any method, including any embodiments disclosed in U.S. Patent No. 8,613,957, entitled “Scaffold Material for Wound Care and / or Other Tissue Healing Applications.” Therefore, as used herein, terms such as “decellularization,” “decellularized fish skin,” and “cell-free fish skin” include fish skin from which a substantial amount of cellular and nucleic acid contents have been removed, leaving the complex three-dimensional interstitial structure of the extracellular matrix material (ECM). Generally speaking, the decellularization described above is a milder form of treatment than other methods required and / or regularly performed on mammalian tissues, often involving harsh chemical treatments and / or storage in chemicals (e.g., antibiotics). 【0199】 The decellularization method described in U.S. Patent No. 8,613,957 results in the creation of a scaffold material that maintains the three-dimensional structure of natural extracellular matrix components, thereby, in some examples, allowing stem cells and other cells contributing to the wound healing process to migrate and / or be supported across the physical medium to promote wound healing. The natural structures of extracellular components such as collagen are maintained within the decellularized fish skin scaffold material, along with other natural components such as omega-3 polyunsaturated fatty acids (PUFAs). 【0200】 Other scaffolding materials derived from mammalian skin / membranes, such as placenta-based wound treatments, may be used as skin substitutes. 【0201】 Skin substitutes based on decellularized fish skin are preferred because there is no risk, or at least a much lower risk, of disease transmission from Atlantic cod (Gadus morhua) and many other fish species to humans. Furthermore, decellularized fish skin tends to be free of allergenic components, significantly reducing the risk of allergies and other immune reactions. Because of the reduced risk of disease transmission and allergic reactions, decellularized fish skin is subjected to gentle processing that preserves the biological structure and bioactive compounds of the extracellular matrix. Thus, although skin cells are removed during processing, decellularized fish skin maintains the natural three-dimensional structure of the extracellular components, providing a natural scaffold that promotes wound healing. In contrast, mammalian scaffold materials lack three-dimensional structure and lose other natural extracellular components, and cannot promote wound healing in the same way or to the same extent as decellularized fish skin. 【0202】 Other forms of collagen-based materials may be used as biological or synthetic skin substitutes, but it is preferable that reconstituted collagen materials are not harvested through harsh physical and chemical treatments that would impair their natural three-dimensional structure, particularly in relation to other natural extracellular components. Similar to the mammalian scaffold materials discussed above, the lack of natural structure and / or the three-dimensional extracellular matrix environment provided by the reconstituted collagen material may reduce the wound-healing effect of the skin substitute. Of course, the selection of a skin substitute must consider the manufacturing cost and consistency of the skin substitute, as well as other factors, and in some cases or applications, the use of such reconstituted collagen materials may indeed be preferable. 【0203】 Additional considerations regarding the onset of infection Wound treatment is often performed by untrained personnel in harsh environments, for example, on or near the site of injury in combat situations. The inventors recognized a critical need for wound treatments with a broad spectrum of antimicrobial activity, possessing tissue regenerative capabilities, bacterial barrier properties, and analgesic properties, such as those found in fish skin grafts. The inventors found that wound treatment products that are easy to store and transport and can function as either final or temporary treatments would be particularly useful, for example, by reducing the need to evacuate wounded individuals in combat or emergencies. 【0204】 As mentioned above, infection is a major challenge in wound management during emergencies and combat. It is a factor in the morbidity and mortality rates of wounded or combat-oriented soldiers. For example, infection accounts for one-third of all casualties, prolongs treatment, and increases the risk of amputation. Due to the unique mechanisms of injury and harsh environments, combat wounds are prone to contamination and more difficult to treat. An early sign of infection is an imbalance of bacteria within the wound. Common pathogens found in early-stage wounds include both Gram-positive (G+) and Gram-negative (G-) strains. Once an infection develops, the emergence of Gram-negative bacteria and multidrug-resistant (MDR) bacteria is observed. Therefore, we have identified a strong need for effective and immediate intervention to reduce the risk of infection and benefit soldiers and emergency responders. 【0205】 The tissue regenerative wound treatment of the present disclosure, which in some embodiments may be a blue antimicrobial fish skin graft, provides a novel visual stimulus for wound healing. The wound treatment of the present disclosure retains the performance benefits of earlier wound treatments, such as grafts that promote wound healing and provide biological coverage for burns, acute and chronic wounds. In addition, the wound treatment of the present disclosure is impregnated with antimicrobial agents, either in the form of antimicrobial colorants such as methylene blue (MB) and gentian violet (GV), or as further additional activators. The wound treatment of the present disclosure integrates into the wound bed over time, releasing antimicrobial agents to prevent the development of infection. The blue color of the skin graft helps reduce unnecessary reapplication, thus minimizing wound exposure and promoting wound healing without permanent discoloration of the peri-wound tissue. 【0206】 Conventional field bandages, available in combat or emergency environments, provide rapid coverage, are deployable on-site even in harsh environments, can be used by the patient themselves or a partner, and can often be used with saline solution for rinsing and dehydration. However, conventional field bandages do not have a wide antibacterial range, need to be changed daily, do not integrate with the wound bed, do not enhance wound healing, and do not provide a visual aid for integration into self-care for others. 【0207】 Antimicrobial silver bandages, which can be used in combat or emergency environments, provide fast-acting coverage, are field-deployable even in harsh environments, can be used by the patient themselves or a partner, and can offer a broad antimicrobial range. However, antimicrobial silver bandages cannot be used with saline for rinsing or rehydration, need to be changed every 1-3 days, do not integrate into the wound bed, do not enhance wound healing, and do not provide a visual aid for integration for self-care or care of others. 【0208】 In comparison, the wound treatment of this disclosure provides a fast-acting covering, is deployable in the field even in harsh environments, can be used by the patient themselves or a partner, and offers a broad antimicrobial range. Furthermore, the wound treatment of this disclosure can be used with saline for rinsing and rehydration, and (based on color visual aids) needs to be replaced after 5-10 days. Significantly, the wound treatment of this disclosure integrates with the wound bed, enhances wound healing, and provides a suitable and effective visual aid for integration of self-care for others. 【0209】 The wound care methods described herein are well suited to combat or emergency environments in order to address the needs of soldiers and medical personnel, for the following reasons: Antimicrobial activity: Methylene blue MB is a potent antimicrobial dye against G- bacteria. It reduces the bacterial burden on wounds and decreases granulation tissue formation. GV is an antimicrobial dye against G+ bacteria and can affect pro-inflammatory mediators; Storage period: The wound treatment is stable at room temperature for more than 3 years and robust against impact. Its stability under long-term high temperature and humidity conditions has been tested. Packaging: In the preferred embodiment, wound treatment is individually packaged in vacuum-sealed military-grade foil pouches containing sheets of dry, sterile fish skin. The pouches are small, lightweight, and easily fit into a pocket or medical bag (100 cm² of fish skin, 2 g). The packaging is resistant to moisture and harsh environments. The product is easy to transport and store and is available in multiple sizes; Ease of Use: Wound care requires basic medical supplies and limited medical knowledge for their use. Colorants help users distinguish between pus / shedding from wound pus and fish skin integrated into the wound bed, enabling clear follow-up treatment. Non-staining: For wound treatment, medical-grade coloring compounds with known staining and degradation profiles are used. No staining was observed with typical topical use. The inventors found that if any pigment was absorbed, the degradation profile was 6–12 days; Removable: There is no need to remove the wound treatment from the wound. Skin substitutes such as fish skin recruit natural human cells into their structure, where the cells eventually convert the skin substitute, such as fish skin, into new tissue. However, if necessary, once the fish skin begins to integrate, the product can be easily removed by lifting it with tweezers or wiping it off with a damp gauze. Use in harsh environments: Wound treatment can be used on or near the injured area as final wound treatment or as a temporary antimicrobial cover. Skin substitutes such as fish skin integrate slowly, resulting in less frequent bandage changes. Pain relief: Wound treatment involves covering the wound with skin and creating an internal environment. In the case of fish skin, the graft is rich in fatty acids, including omega-3, which helps shield exposed nerve endings, reduce inflammation, and positively impact pain through lipid mediators. 【0210】 A key objective of this disclosure is to provide the Department of Defense and emergency responders with an innovative solution for wound management at or near the site of injury as an FDA-approved antimicrobial skin substitute. The wound treatment of this disclosure offers superior healing properties along with potent antimicrobial activity. The wound treatment can be applied as final care for smaller, less severe wounds and as a temporary antimicrobial cover for more severe injuries requiring a transition to higher-level care. Furthermore, the colorant helps healthcare providers distinguish the skin substitute being integrated from pus or wound shedding tissue. 【0211】 The wound treatments of this disclosure promote wound healing through a combination of the following approaches: 1. Acting as an extracellular matrix that integrates into the wound, providing structural support for host cells to heal and regenerate tissue. 2. MB and GV inhibit G+ and G- bacteria along with fungi, thus preventing biofilm formation and reducing the risk of infection. 3. Fewer bandage changes result in less wound exposure to contamination and less mechanical trauma from repeated bandage removal. 4. Color serves as a guide for untrained users in optimal bandaging and antimicrobial management. 5. Biomolecules naturally present in skin substitutes, such as fish skin (omega-3 and collagen), or additional activators reduce pain, inflammation, and bleeding. 【0212】 The wound treatments described herein provide final and temporary treatment for minor and severe wounds / burns by preventing infection, providing coverage, and promoting healing. 【0213】 Preferred embodiments of skin substitutes, such as decellularized and freeze-dried fish skin grafts, are highly effective in initiating and promoting the natural healing process. Skin substitutes, and in particular physical scaffolds, and even more preferably fish skin physical scaffolds, provide biomolecules that enable cell infiltration and reduce inflammation and pain. These properties have been demonstrated numerous times in in vitro, in vivo, and clinical studies. In addition, in a more preferred embodiment, fish skin is rich in natural omega-3 fatty acids, which have been shown to act as a barrier against bacterial invasion, with potential antiviral, bacteriostatic, and antimicrobial effects. 【0214】 A preferred embodiment of fish skin provides bacterial barrier properties. Perhaps the most compelling evidence for the ability of unstained fish skin to mitigate wound infection is a study conducted at the Curie Institute in Paris involving 21 independent patients, where the infection rate at split-thickness donor sites decreased from 60% to 0% for wounds treated with fish skin. Even unstained fish skin grafts can act as a bacterial barrier against Staphylococcus aureus for up to 48–72 hours under optimal bacterial growth conditions. In vivo studies on infected mouse models have demonstrated that fish skin can act as a bacterial barrier against Proteus mirabilis (P. mirabilis), one of the most frequently identified MDR strains in combat trauma-related infections. 【0215】 Methylene blue and gentian violet even offer additional antimicrobial properties. Advances in wound care have resulted in combinations of antimicrobial agents such as silver, iodine, and polyhexamethylene biguanide (PHMB) with conventional wound dressings. While silver and iodine exhibit robust antimicrobial activity, prolonged use of these agents leads to high levels of cytotoxicity to host cells. MB and GV are FDA-approved, can be used topically, and have demonstrated excellent efficacy in managing chronic wounds with local infections. 【0216】 In vitro data show promising results for prototypes of preferred embodiments of this disclosure. The tests were based on ASTM E2149 and Kirby-Bauer Zone of Inhibition assays. Both assays showed that fish skin grafts impregnated with MB and GV efficiently inhibited both Escherichia coli (E. coli) and Staphylococcus aureus in solution and on agar plates. 【0217】 Figures 14A and 14B show (A) the results of the ASTM E2149 assay against Escherichia coli, and Figure 14C shows (B) the results of the Kirby-Bauer Zone of Inhibition assay against Staphylococcus aureus. Figures 14A and 14B show the results of placing antimicrobial fish skin in a suspension of Escherichia coli and shaking it for up to 24 hours, and a clear reduction in bacteria was observed between the antimicrobial fish skin (Disc 1) (Figure 14A) and the original fish skin (Disc 2) (Figure 14B). In Figure 14C, (B) fish skin treated with different concentrations of methylene blue and gentian violet in the range of 0.1% w / v (Section 1410), 0.5% w / v (Section 1420), and 1% w / v (Section 1430) showed clear inhibition regions on agar plates inoculated with Staphylococcus aureus, while no inhibition regions were observed in the original fish skin. 【0218】 The applicant possesses a wealth of scientific data demonstrating the healing properties of fish skin. This includes two randomized clinical trials on acute wounds that showed fish skin resulted in more effective healing compared to mammalian cell and tissue-based products (CTPs), such as (Oasis)17 and human amniotic / chorionic membrane in terms of complete healing time, and a study of clinical donor sites in which the use of fish skin halved the healing time of patients. There are numerous independent case series publications that have yielded overwhelmingly positive results regarding fish skin as an exemplary and preferred embodiment. 【0219】 The production of tissue-regenerating wound treatments, and especially fish-skin-based tissue-regenerating wound treatments, is highly feasible. The additional step of impregnating the skin substitute with an antimicrobial color would require minimal additional equipment. MB and GV are readily available in formulation quality grades. 【0220】 Embodiments of tissue-regenerative wound treatments of this disclosure, and in particular embodiments comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, may be effectively used as transient antimicrobial scaffolds for the management of wounds including diabetic foot ulcers, arterial ulcers, pressure ulcers, venous leg ulcers, and traumatic ulcers. Combinations of these types of wounds are thought to account for 54% of lower limb amputations in the United States, which is an irreversible state of debilitation. Nearly half of those who undergo amputation due to vascular disease die within five years. This is higher than the five-year mortality rates for breast cancer, colon cancer, and prostate cancer. 【0221】 The standard care in the United States for the treatment of chronic ulcers is as follows: Established usual or standard care for chronic wounds incorporates common principles that apply to the management of all types of wounds, as follows: remove necrotic tissue by debridement (typically with a sharp debridement); maintain fluid balance by selecting an appropriate wound dressing to control exudate; take measures to prevent or treat wound infection; correct ischemia in the wound area; apply some form of compression for venous leg ulcers; and bring about some form of offloading for diabetic foot ulcers. 【0222】 Embodiments of tissue regenerative wound treatments of the present disclosure, and in particular embodiments comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, may provide a more effective treatment for chronic wounds compared to established SOCs for skin substitutes, as they are more effective as treatments for chronic wounds by resulting in the formation of a temporary scaffold within the bandage and resisting bacterial growth compared to SOCs. For the purposes of this application, it is expected that standard care will be defined in the same manner as the definition of the Agency for Healthcare Research and Quality (AHRQ). Decellularized fish skin wound products of predicate devices have been shown in randomized clinical trials to promote significantly faster healing compared to standard care collagen bandages. The devices provide a more effective treatment compared to current standard care as defined by the AHRQ. 【0223】 Embodiments of tissue regenerative wound treatment of the present disclosure, and in particular embodiments comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, achieve the same improvements compared to SOC, while also providing resistance to bacterial growth and possessing distinct properties as a temporary scaffold. 【0224】 A preferred embodiment of the tissue regenerative wound treatment of this disclosure includes fish skin as a skin substitute, but other skin substitutes different from fish skin products or fish skin-based wound treatments provided by Kerecis® may, of course, be used. 【0225】 For extended comparison, the device in question provides a more effective treatment compared to emerging treatments. This application includes emerging treatments that have been given Q codes as skin substitutes under the Common Healthcare Therapeutic Procedures Code System (HCPCS). 【0226】 As described above, the group of skin substitutes that can be used as examples of skin substitutes according to this disclosure is large and diverse. The AHRQ Technical Brief Project ID WNDT0818, "Skin Substitutes for Treating Chronic Wounds," published on February 2, 2020, identifies 76 commercially available products in Table 2 on pages 9-13, but there are few studies comparing them in-house. Each of these listed skin substitutes could be an embodiment of a skin substitute according to this disclosure. 【0227】 Arguments for more effective treatments focus on comparing treatment outcomes, antimicrobial properties, temporary scaffolding properties, and their impact on usability. 【0228】 The combination of antimicrobial colors for biodegradable scaffolding should not interfere with each other's basic functions, and may even produce a synergistic additive effect. 【0229】 Temporary scaffold formation can be understood as a tissue scaffold that aids tissue regeneration by supporting inward cell growth, angiogenesis, and the regeneration of the extracellular matrix. Current temporary scaffolds cannot prevent bacterial growth. In fact, in some cases, collagen can act as a nutrient for bacteria. Current temporary scaffold products are not suitable for use in wound care. 【0230】 Antimicrobial products prevent bacterial colonization of devices, but they do not necessarily help in scaffolding formation (in the case of silver-based products, they can actually be harmful due to their cytotoxic effects). 【0231】 The tissue regenerative wound treatments of this disclosure, and in particular those comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, provide what may be called “device identification.” When absorbable bandages are applied to a wound, it can be difficult to identify what is an active but partially absorbed device or what is wound shedding tissue that should be removed. This can lead to premature bandage changes. Currently, there are no absorbable wound products that can identify by color. 【0232】 The combination of scaffolding and antibacterial colors creates a synergistic additive effect. 【0233】 The tissue regenerative wound treatment of this disclosure, and in particular the tissue regenerative wound treatment comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, is the first wound care product known to the inventors that provides temporary scaffold formation and device identification of absorbable wound dressings. Combined with antimicrobial protection, the risk of bacteria causing inflammation or multiplying within the product and accelerating degradation is limited. Furthermore, easy device identification allows for more accurate dressing changes. 【0234】 Temporary scaffold formation can be compared to other skin substitutes. Temporary scaffold formation supports intrinsic cell growth, angiogenesis, and extracellular matrix regeneration. As the field of tissue engineering continues to evolve, the criteria for ideal skin grafts have shifted to materials that support cell integration and tissue proliferation. These criteria include the need for the scaffold to meet one or more, preferably all, of the following: enable and promote intrinsic cell growth; allow for uniform spatial distribution of cells; support extracellular matrix regeneration; support angiogenesis; do not cause foreign body type reactions; integrate rapidly into the wound; and be mechanically strong and stable. 【0235】 The inventors have shown that the fish skin grafting technique as described in the present disclosure can provide a temporary scaffolding function. Further, evidence has been shown that the results of scaffolding formation on cell ingrowth, angiogenesis, and extracellular matrix regeneration are more effective than other known devices such as absorbent collagen devices, for example Primatrix. 【0236】 Based on these results, there is evidence that the tissue regeneration wound treatment of the present disclosure, and particularly the tissue regeneration wound treatment that includes fish skin as a skin substitute and provides antibacterial properties by either (one or more) colorants or further additional active agents, results in a treatment that is more effective than standard care by functioning as a temporary scaffold. 【0237】 By adding antibacterial agents to the original fish skin, antibacterial protection is provided to the device. The inventors have also discovered that the addition of a suitable colorant does not interfere with the basic scaffolding effect of fish skin. MB and GV are organic dyes that can be used to reduce microorganisms in the clinical setting while minimizing toxicity to humans. MB and GV can be used topically for the rapid management of the local bacterial load within the wound. The concentrations of MB and GV in the preferred embodiments are controlled to be below 0.00025 g / g (0.01%), lower than the concentration of Hydrofera Blue Ready (below 0.0035 g / g for each color) and significantly lower than the concentrations of 1% MB and GV in commercially available topical agents. Of course, Hydrofera Blue Ready may be used as another embodiment of the colorant. GV and MB can be used in combination with enzymatic debriding agents, growth factors, and hydrogels without inhibiting the action of companion products. 【0238】 MB and GV used in the tissue regeneration wound treatment of the present disclosure, particularly the tissue regeneration wound treatment including fish skin, have been discovered by the inventors not to impair the scaffold-forming effect of fish skin. The addition of MB or GV to fish skin can be carried out at the final stage of the manufacturing process before sterilization. In this process, the original design, material, function, packaging, and sterilization of the fish skin are not changed. 【0239】 In a recent study (Stone II, International Journal of Molecular Sciences, 2021), a comparison between fish skin grafts and fetal bovine dermis (Primatrix) was conducted when treating deep partial thickness (DPT) burns in a preclinical pig model. The purpose of this study was to determine how effective fish skin grafts are for DPT burns, how they integrate, and whether they improve long-term healing. Under the study conditions, it was found that fish skin grafts integrate into the wound bed faster than fetal bovine dermis. As a result of the fish skin grafts, re-epithelialization became faster starting from the 10th day and until the 28th day, particularly on the 14th day, and the difference between the fish skin grafts and fetal bovine dermis was significant. As a result of the fish skin grafts, blood flow increased and newly formed blood vessels increased. The fish skin grafts promoted the complete formation of the epidermis after 21 days. Also, the fish skin grafts hardly caused an inflammatory reaction (few foreign bodies and few inflammatory cells). 【0240】 The results of this study provide evidence that fish skin grafts are a preferred embodiment, but fetal bovine dermis (Primatrix) products can, of course, still be used as an effective skin substitute in accordance with the present disclosure and may also be a preferred embodiment of the skin substitute as contemplated in the present disclosure under some conditions or considerations. 【0241】 Furthermore, although this study was conducted with uncolored versions of fish skin products without the antimicrobial agents MB and GV, the extent of the resulting wounds was classified as deep, partial-thickness burns that damaged both the epidermis and dermis and were often complex and time-consuming to treat. The role of fish skin in this study is not only as a temporary covering but also as a temporary scaffold for long-term healing. This study provides many important insights into the scaffold-forming effect of the original fish skin. 【0242】 The tissue regenerative wound treatment of this disclosure, and in particular the tissue regenerative wound treatment comprising fish skin as a skin substitute, wherein antimicrobial properties are provided by either (one or more) colorants or further additional activators, prevents bacterial colonization of the target device compared to other skin substitutes currently known. 【0243】 The term "bacterial barrier" can be understood to mean that broad-spectrum antimicrobial agents provide a barrier against bacterial invasion of the bandage, as this can help reduce infection and ensure that the temporary scaffold functions as intended. 【0244】 In a broad sense, skin substitutes can be considered biodegradable tissues that are infiltrated by the body's own cells and subsequently integrated, absorbed, or broken down. Most skin substitutes have a poor inherent ability to defend against bacterial invasion and can colonize if bacteria are present in the wound. Bacterial colonization of skin substitutes accelerates their degradation and makes intrinsic growth of host cells less likely. 【0245】 Of the 76 skin substitutes, two other skin substitutes that provide some antimicrobial effects, namely PriMatrix AG and PuraplyAM, are listed. However, neither of these two was found to have the same antimicrobial spectrum and antimicrobial activity as the antimicrobial agents used in the tissue regenerative wound treatment of this disclosure, and in particular fish skin as a skin substitute, with antimicrobial properties provided by either (one or more) colorants or further additional activators. Of course, as stated above, PriMatrix AG and PuraplyAM can still be considered as embodiments of skin substitutes in this disclosure, and in fact, may be preferred embodiments in certain circumstances and conditions. 【0246】 [Table 2] TIFF0007875881000007.tif246163 【0247】 Of course, as stated above, PriMatrix AG and PuraplyAM can still be considered embodiments of skin substitutes in this disclosure, and in fact, may be preferred embodiments in certain situations and conditions. The antimicrobial range of the devices in question is comparable to that of Hydrofera Blue, a bandage with equivalent MB and GV concentrations. 【0248】 The tissue regenerative wound treatments of this disclosure, and in particular those comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, may provide what may be termed “device identification” that facilitates an optimal utilization cycle. “Device identification” may be understood as a colorant that facilitates product identification when integrated into a wound bed. 【0249】 Skin substitutes are most transparent or off-white before application and become transparent, white, or caramel-colored upon integration into the wound bed. This appearance can sometimes be indistinguishable from wound slough, exudate, or biofilm, especially for inexperienced users. This makes it difficult to determine whether the skin substitute has fully integrated and needs replacement, or whether it is still partially active and can be retained in the wound longer. Failure to determine whether there is still active skin substitute in the wound can lead to three consequences: (1) mistaking slough for collagen bandages, leading healthcare providers to fail to remove the slough from the wound, thus delaying wound healing and increasing the risk of infection; (2) mistaking active products in the wound for slough, leading healthcare providers to prematurely remove the device; and (3) removal of the transient active scaffold tissue accompanied by inward growth of fresh host cells. 【0250】 If active products within a wound are mistaken for sloughed-off tissue, it can lead to premature reapplication of the device, resulting in unnecessary interventions and associated costs for the patient. 【0251】 Novel devices such as those disclosed herein are colored using biocompatible colorants that safely and easily distinguish them from detached tissue or other tissues. This is done using disclosed colorants that bind color to the graft. 【0252】 This device represents a groundbreaking technology and a novel application of innovative technology that could lead to clinical improvements in the treatment of chronic, non-healing wounds and the prevention of potential amputations. The device provides a 3D structure that supports human cell invasion and proliferation, as well as angiogenesis, while inhibiting bacterial colonization on the scaffold. 【0253】 The tissue regenerative wound treatments of this disclosure, and in particular those comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, have at least one or more, preferably all, of the following features: providing a stable, absorbable scaffold that promotes intrinsic cell growth and angiogenesis; providing a broad-spectrum coverage to address microorganisms often present in wounds; not causing toxicity to host cells or inhibiting intrinsic cell growth compared to silver-containing bandages; and not causing mutations in bacterial antimicrobial resistance compared to antimicrobial bandages. 【0254】 Furthermore, depletion of the coloring agent can cause a change in the color of the bandage, which can provide an important visual indicator for guiding the bandage's replacement. 【0255】 The applicant possesses multiple in vitro and in vivo transient scaffold data for the previously defined Omega3 Wound device. The inventors' evidence indicates that the addition of a coloring agent (antimicrobial agent) to the scaffold does not interfere with the basic function and has a synergistic additive effect. 【0256】 In an in vitro study on intrinsic cell growth (Magnusson, Military Medicine, 2017), it was found that fibroblasts infiltrated and reconstructed the fish skin graft after 12 days compared to hHACM material with less fibroblast infiltration. The tissue regenerative wound treatment of this disclosure, and in particular the tissue regenerative wound treatment comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, retains the same porous structure and pore size as the original fish skin that induces cell infiltration into the scaffold. To address the toxicity of MB and GV to cells, the inventors conducted preliminary cytotoxicity tests and found that MB and GV do not pose a cytotoxicity concern. Furthermore, since the reference device, Hydrofera Blue Ready, contains higher concentrations of MB and GV but did not cause cytotoxicity concerns, the tissue regenerative wound treatment of this disclosure, and in particular the tissue regenerative wound treatment comprising fish skin as a skin substitute and provided with antimicrobial properties by either (one or more) colorants or further additional activators, should not cause any adverse effects on intrinsic cell growth. 【0257】 Preliminary bench studies have demonstrated the efficacy of the tissue-regenerating wound treatments of this disclosure, particularly those containing fish skin as a skin substitute and having antimicrobial properties provided by one or more colorants (MB and GV), against antimicrobial activity. These studies were conducted using three of the most common microorganisms in wound infections: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The testing methods ranged from simple, basic assays such as agar disc diffusion to more challenging industrial standard tests such as AATCC1OO and ASTM E2149. Agar disc diffusion results showed that the tissue-regenerating wound treatments of this disclosure, containing fish skin as a skin substitute and having antimicrobial properties provided by one or more colorants (MB and GV), formed a distinct inhibitory region against Staphylococcus aureus, compared to Omega3 Wound and Primatrix AG. The diameter of the inhibitory area was 17.25 + 0.5 mm with the tissue regenerative wound treatment of this disclosure, which includes fish skin as a skin substitute and is provided with antimicrobial properties by one or more colorants (MB and GV), and 11.67 + 0.58 mm with Primatrix AG. However, since fish skin did not show any effect, it had an inhibitory zone with the same diameter as the sample (6 mm). The results of the AATCC1OO evaluation demonstrated the high efficacy of the tissue regenerative wound treatment of this disclosure, which includes fish skin as a skin substitute and is provided with antimicrobial properties by one or more colorants (MB and GV), against Staphylococcus aureus and Pseudomonas aeruginosa. For Pseudomonas aeruginosa, the reduction rate was estimated to be approximately 98%. The tissue regenerative wound treatment of this disclosure, which includes fish skin as a skin substitute and is provided with antimicrobial properties by one or more colorants (MB and GV), showed potent antimicrobial efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa. The results of the ASTM E2149 test demonstrated a reduction in growth in E. coli suspension by Kroma Antivirus. Using the tissue regeneration wound treatment of this disclosure, which included fish skin as a skin substitute and provided antimicrobial properties by one or more colorants (MB and GV), 37 colonies were formed on an agar plate, compared to 445 and 491 colonies on uncolored fish skin and the E. coli suspension itself.In the benefit of the tissue regeneration wound treatment of the present disclosure, which includes fish skin as a skin substitute and the antibacterial property is provided by any of the (one or more) colorants (MB and GV), the growth reduction rate was approximately 92 - 93%. 【0258】 Considering the promising results of the inventors from our preliminary tests, the tissue regeneration wound treatment of the present disclosure, which includes fish skin as a skin substitute and the antibacterial property is provided by any of the (one or more) colorants (MB and GV), provides a more effective antibacterial treatment for various types of wounds. 【0259】 Collagen scaffolds are widely used in chronic wound management to enhance the wound healing process. Bioactive wound dressings have advantages over other types of dressings because they are biocompatible and enhance intracellular growth and tissue regeneration due to properties such as an EMC template. 【0260】 The inventors have discovered and disclosed various embodiments, including a preferred embodiment that uses a temporary scaffold of fish skin combined with two antibacterial colorants for wound management as an effective barrier against the colonization of microorganisms in the scaffold. The temporary scaffold supports angiogenesis and intracellular growth while inhibiting the colonization of microorganisms in the dressing. 【0261】 A preferred embodiment of the tissue regenerating wound treatment of this disclosure, comprising fish skin as a skin substitute and provided with antimicrobial properties by one or more colorants (MB and GV), is a cell-free absorbable fish skin wound matrix. The wound treatment acts as a temporary scaffold that supports angiogenesis and intrinsic cell growth while inhibiting bacterial colonization on the scaffold. The device comprises two antimicrobial agents that provide broad-spectrum antimicrobial protection on the scaffold with methylene blue and gentian violet (crystal violet). The device is supplied as a sterile, intact, or mesh sheet up to 20 × 30 cm in size. The broad-spectrum antimicrobial agents provide a barrier against bacterial invasion of the bandage, as they can help reduce infection and ensure that the temporary scaffold functions as intended. 【0262】 Appropriate Use In preferred embodiments, the tissue regenerative wound treatment is intended as an antimicrobial temporary scaffold for the management of wounds including diabetic foot ulcers, arterial ulcers, pressure ulcers, venous leg ulcers, and traumatic wounds. 【0263】 Device Configuration A preferred embodiment of the tissue regeneration wound treatment is a fish skin medical device for wound management. The subject scaffold material, sometimes referred to hereafter as the device, is obtained from the skin of wild North Atlantic cod (Gadus morhua) by a normalized and controlled manufacturing process and is supplied in final sterile packaging of skin pouches in the following sizes: 16 mm disc; 2 × 2 cm; 2 × 4 cm; 5 × 5 cm; 10 × 10 cm; 20 × 30 cm. The device may also be supplied in a particle form, as described and shown above. 【0264】 The device may contain two antimicrobial agents, such as methylene blue and gentian violet (crystal violet), to provide broad-spectrum antimicrobial protection on the scaffold. The concentrations of MB and GV are controlled to 0.00025 g / g (0.01%) or less, but may be as low as 0.1%. 【0265】 The target device preferably integrates completely with the surrounding tissue over time in response to the deposition of new host tissue. The desirable physical properties of the target device enable intrinsic cell growth. The target device is preferably biocompatible, non-crosslinked, bioabsorbable, strong, and flexible. Its tensile strength supports fixation by sutures or staples. 【0266】 The operating mechanism of the target device can be broken down into the following three main areas: 1. Collagen dressing: substantially equivalent to the decellularized fish skin wound product (K132343) and possessing the following differentiated characteristics: 1.1. Impregnated with antimicrobial coloring agent: 2a: "Bacterial barrier": The broad-spectrum antimicrobial agent provides a barrier to bacterial invasion of the dressing, as it can help reduce infection and ensure that the temporary scaffold functions as intended. 2b: "Device identification": The coloring agent facilitates product identification when integrated into the wound bed. 2. "Temporary scaffold formation": Temporary scaffold formation that supports inward cell growth, angiogenesis, and regeneration of the extracellular matrix. 2.1. Collagen dressing. The target device functions substantially equivalent to the decellularized fish skin wound product as a collagen dressing with the same basic operating mechanism. 【0267】 The primary purpose of collagen scaffolds is to act as templates that mimic the extracellular matrix (EMC) of healthy tissue. By mimicking and supporting cells, they assist in the reconstruction of many different types of tissue, thus aiding the wound healing process. Each component of the EMC is essential for each stage of wound healing. Components of the EMC play a crucial role in assisting cell proliferation and differentiation, inducing cell migration, and regulating cellular responses. Exogenous EMC is broken down and replaced by natural collagen through the natural remodeling of healthy tissue at the wound site. Decellularized fish skin wound products re-establish functional EMC in chronic wounds. Collagen bandages provide: (1) a moist wound environment, (2) fluid management, and (3) control of fluid evaporation. 【0268】 Decellularized fish skin wound products are substantially equivalent to many porcine collagen matrices approved through the 510k premarket notification process and can therefore be used as collagen bandages. The effectiveness of these devices as collagen bandages for wound management has been demonstrated by a non-inferiority trial compared to Oasis Wound Matrix, a mammalian-derived collagen bandage. The trial concluded that the fish collagen bandage was no less effective than the porcine-derived collagen bandage, no side effects were identified, and improved wound healing was observed over a 28-week period. 【0269】 The only technical difference between decellularized fish skin wound products and the target device is the addition of colorants. There is no evidence or literature evidence that either colorant disrupts or crosslinks the collagen scaffold. Therefore, based on our evidence, there is sufficient evidence to suggest that the target device, whether it is a fish skin-based skin substitute or another skin substitute, also acts as a scaffold mimicking the extracellular matrix to support intrinsic cell growth and angiogenesis. 【0270】 The colorants of the preferred embodiment have a significant antimicrobial effect. Using a colorant of approximately 0.01% mixture of methylene blue (MB) and gentian violet (GV), the two antimicrobial agents provide broad-spectrum antimicrobial protection against both Gram-negative and Gram-positive bacteria. When MB and GV in the target device come into contact with bacteria, they eliminate the bacteria by making bacterial growth in the device unsustainable. 【0271】 Methylene blue is part of the phenothiazine family and was one of the first FDA-approved treatments for malaria when resistance to antimalarial drugs developed. MB has demonstrated its bacterial inactivation in vitro with a wide range of microorganisms, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. 【0272】 The pigments in MB and GV serve as indicators to distinguish between the device and detached tissue within the wound. Since the skin substitute is an absorbable bandage, the color informs the physician when the bandage has fully integrated and a second application is needed. The color also helps reduce the risk of accidentally removing the bandage when performing debridement on the wound. Uncolored collagen bandages can sometimes be difficult to distinguish from detached tissue when partially integrated. 【0273】 For example, Figure 15A shows a graft in a bacterial-filled wound that has turned into sloughed-off tissue. For comparison, Figure 15B shows a fish skin graft in a wound that is approximately 50% integrated and should remain in the wound. However, as can be seen by comparing the sloughed-off tissue in Figure 15A with the graft in Figure 15B, it is difficult to accurately and easily distinguish between a graft that has undergone inward growth and a graft that has become sloughed-off tissue. For comparison, Figure 15C shows a skin substitute, in this case a fish skin graft, colored with MB / GV colorant. This is evident from the fact that the graft in Figure 15C is integrated, has undergone inward growth, and should remain in the wound for another week and should not be removed. 【0274】 In an in vitro study, mouse embryonic fibroblasts were added to the top of fish skin, demonstrating that the skin scaffold is highly porous, allowing cells to migrate and proliferate within the scaffold. In animal studies, fish skin was applied to burns in a pig model. Fish skin grafts resulted in accelerated wound healing and increased newly formed blood vessels, in addition to superior blood flow beneath the fish skin. In the same pig study, fish skin grafts promoted complete epidermal formation after 21 days, resulting in faster re-epithelialization and a less inflammatory response. 【0275】 When applied to a patient's wound, the embodiment of the target device rapidly integrates into the wound, providing a temporary scaffold for cell migration and proliferation, while MB and GV molecules inhibit and remove microbial colonization on the matrix. The abundant dermal collagen fibers support intrinsic cell growth, angiogenesis, and extracellular matrix regeneration, which are crucial for accelerating wound healing. 【0276】 Colored skin substitutes, such as fish skin grafts, are eventually broken down in the body. Ultimately, intracellular growth of primary fibroblasts containing some inflammatory components completely remodels and breaks down the original skin substitute, such as fish skin grafts, and its color. 【0277】 The enzymatic process primarily involves the hydrolysis of collagen into smaller, easier-to-process particles and the reduction of colorants. 【0278】 Colored skin substitutes, such as fish skin grafts, may have dimensions up to 7 × 20 cm, or even 20 × 40 cm, and may be solid or mesh-like, and may be in sheet or granular form. 【0279】 Further examples of wound treatment generation In yet another example of a method or procedure for generating an embodiment of tissue regeneration wound treatment, the following procedure was followed. 【0280】 Before our arrival, the 10 skins were packed and frozen flat. 【0281】 The solution was prepared by mixing colored solvents containing 0.01% and 0.005% dilutions of MB&GV. 【0282】 To obtain clean water, we used a sink, monitored the bacterial count, and boiled it before use. 【0283】 First, a 1% stock solution was prepared. 【0284】 GV: 650mg pharmaceutical grade (USP), SA-1290002, LOT G1K417, SP1098511 (Distica) 【0285】 MB: Hydrated methylene blue for microscopy, ≥97%.0%, Sigma-AIdrich 66720-100g, LOT #BCBZ4929 【0286】 0.4g of MB and 0.4g of GV were added to 40ml of clean (still warm) water in a boiled flask. Note that 250mg of GV remains. 【0287】 Two 2L bottles were filled with 2L of clean / boiled water (measured by weight). 10ml of water was removed from one bottle using a clean pipette, and 20ml of water was removed from the other bottle. These volumes were replaced with stock solution to prepare 0.005% and 0.01% MB and GV solutions, respectively. After preparing the final solutions, the water in the bottles was quite hot to the touch, which could have affected the staining results. 【0288】 It should be noted that the two compounds stained all surfaces extensively, which meant that a thorough cleaning of all surfaces using water and ethanol was necessary. 【0289】 The fish skin was then removed from the freezer, placed on ice, and transported along with the coloring solution. Everything was brought into a high-risk chamber next to the freeze-dryer. The fish skin was thawed under running tap water from the faucet in the high-risk chamber. Because the fish skin was quite large and long, once it became soft and pliable, it was cut into two shorter pieces. Five pieces of fish skin (divided into 10 equal parts) were placed on two aluminum trays for staining. 【0290】 Approximately 700 ml of Kroma solution was placed in each tray and labeled to 0.01% and 0.005%, respectively. The two trays were then placed in a plastic bag, folded to reduce the risk of spillage, and left on a shaker set at 30 rpm for 2 hours. 【0291】 After approximately 20-30 minutes, the fish skins were moved with sterile wipers to promote uniform staining. After approximately 20 minutes, it was evident that the staining solution had decreased in density and become clear as the fish skins absorbed the stain. To compensate for this, approximately 300 ml of staining solution was added to each tray, meaning the final stained volume was approximately 1000 ml. 【0292】 A portion of both the original coloring solution and the remaining staining solution used was stored in 50 ml tubes to allow for later quantification of concentration. This, combined with measurements of the size and weight of the fish skin after freeze-drying, allowed for a rough quantification of color absorption into the fish skin. 【0293】 Since it takes about 45 minutes to prepare for execution, we started using the freeze dryer immediately after 18:00. 【0294】 A computer error made it difficult to start the freeze-dryer. Therefore, staining took approximately 3 hours (note that the shaker returned to a faster default shaking speed after 3 hours). The skin was thoroughly washed with running tap water for 10-15 minutes in a high-risk room. 【0295】 The remaining coloring solution had clearly become somewhat clearer again, and there was a slight color difference between the two batches of fish skin: 0.01% was true denim dark blue, while 0.005% was closer to denim blue. Where quantification was possible, samples of the remaining staining solution were collected in 50 ml tubes. Approximately one and a half plates were needed for both batches to have a total of three complete plates. The more strongly stained skin was on the left-hand side of the shared plate. 【0296】 I started the freeze-drying process around 8:30 p.m. and left it overnight. 【0297】 The fish skin was freeze-dried in the morning and packaged for non-sterile use. Another batch was repeated the following day using the remaining 0.01% and 0.005% solutions (at room temperature). 【0298】 Further examples of generating prototype wound treatments Two prototypes of colored cod skin are produced at two different concentrations of methylene blue and gentian violet. The concentrations of the color solutions are 0.01% w / v aqueous solution w / v; methylene blue (50%) and gentian violet (50%), and 0.005% w / v aqueous solution w / v; methylene blue (50%) and gentian violet (50%). 【0299】 Materials: 10 descaled and decellularized cod skins, Batch DC 21039A; 1 liter of 0.01% w / v aqueous solution; 50% methylene blue and 50% gentian violet; 1 liter of 0.005% w / v aqueous solution; 50% methylene blue and 50% gentian violet; 10 aluminum trays; scissors; small Tyvek pouches; large Tyvek pouches; large plastic bags; shaker; sealer. 【0300】 Prototype process: All cod skins were fresh, from those produced on the same day. They were frozen at -80°C for 5 hours. The cod skins were too large to fit in the aluminum trays, so they were cut in half, resulting in a total of 20 pieces of cod skin. 【0301】 Prototype 0.01% One liter of 0.01% solution was poured into an aluminum tray marked MB-GV 0.01%, and ten cod fillets were placed evenly on the tray to ensure the solution completely covered the skin. The tray was placed in a plastic bag to minimize the risk of color spillage, and then the tray was left on a shaker with pro:40 for three hours. 【0302】 Prototype 0.005% One liter of 0.005% solution was poured into an aluminum tray marked MB-GV 0.005%, and ten cod fillets were placed evenly on the tray to ensure the solution completely covered the skin. The tray was placed in a plastic bag to minimize the risk of color spillage, and then the tray was left on a shaker with pro:40 for three and a half hours. 【0303】 Start of coloring on shaker: 15:40 ± 10 minutes 【0304】 Coloring on the shaker completed: 19:05 ± 5 minutes 【0305】 Rinse start time: 19:05 ± 5 minutes 【0306】 Rinse completion time: 19.20 ± 5 minutes 【0307】 Freeze-drying: All fish skins were spread on a steel plate, and another plate was placed on top to sandwich them. Freeze-drying machine program: SvavaColor - 10 hours total time. 【0308】 Packaging: Clean, dry, and colored cod skin was supported by visual inspection and bending tests to prepare it for packaging. The samples were packaged into Tyvek pouches, then into large and small sample pouches, marked, and sealed. 【0309】 No skin removal was performed on these prototypes. 【0310】 Crosslinking to improve the stain fastness and mechanical properties of wound treatments. In further embodiments, the inventors have found that crosslinking of skin substitutes, such as scaffolding materials, can further enhance the properties of the skin substitutes, including increasing the fastness of colorants used to color the skin substitutes, improving the mechanical material properties of the skin substitutes, increasing the resistance of the skin substitutes to enzymatic and chemical degradation, and extending the lifespan of colorants added to the skin substitutes under biological conditions such as treated wounds. In preferred embodiments, the primary objective of crosslinking skin substitutes, such as scaffolding materials, is to obtain a colored product that maintains its color for at least one day, preferably three days, after application to a wound, and more preferably up to eight to ten days, and even more preferably up to fourteen days, after application to a wound. 【0311】 As described herein, crosslinking of skin substitutes and / or skin substitutes to which colorants have been added can be carried out by various means, such as irradiation or chemical means. 【0312】 Chemical crosslinking or modifying factors In one embodiment, the skin substitute is crosslinked by treatment with a crosslinking agent. In another embodiment, the proteins of the skin substitute are further modified by treatment with a protein modifier. 【0313】 In embodiments, the chemical crosslinking agent targets one or more of the following groups: primary amines (-NH2); carboxyls (-COOH); sulfhydryls (-SH) or carbonyls (-CHO), or other groups. Thus, the crosslinking agent may be, for example, amine-reactive, carboxyl-to-amine-reactive, sulfhydryl-reactive, and / or aldehyde-reactive. 【0314】 In the first embodiment, the crosslinking agent is a monosaccharide or monosaccharide. Alternative sugars, including glucose, fructose, and galactose, may be used. For example, the crosslinking agent may be ribose or contain ribose. Alternative sugars, including glucose, fructose, and galactose, may be used. Oligosaccharides and disaccharides may also be considered. 【0315】 In another embodiment, the crosslinking agent is a natural or synthetic crosslinking agent. For example, in the embodiment, the crosslinking agent contains or is genipine. 【0316】 Example 1 - Ribose Crosslinking A first example of the use of ribose as a crosslinking agent is provided herein. 【0317】 According to this embodiment, a standard ribose (stock) solution is prepared. For this purpose, a 0.2 M (molar) solution of ribose was prepared in PBS containing 0.05% (w / v) sodium azide to prevent bacterial growth. Other concentrations of ribose or other bacterial growth inhibitors may be used. In this embodiment, the solution contains 30.03 g by weight of ribose, 9.55 g of pre-mixed PBS standard, and 50 mg of sodium azide. The dry components are weighed using a precision balance and added to a 1 L volumetric flask, and diluted with deionized water until the volume reaches 1.00 L. The mixture is stirred until the components are completely dissolved, and then the solution is prepared for use. 【0318】 In this example, Kerecis® fish skin-derived cell scaffold products are used as skin substitutes. Generally, any size and / or number of scaffolds, including even granulated scaffold material, could be processed. The container in which the scaffold material is added to the solution may be large, and the volume of the ribose solution completely covers the sample. In this example, the scaffold material was 4 × 8 cm 2The fish was cut into fragments. Five fragments or samples were cut from a larger sample so that the longer side (8 cm) was parallel to the length of the cod skin. The samples were then immersed in approximately 250 mL of ribose solution at room temperature for 3–6 days. The first sample was removed at the mark on day 3, the next two on day 5, and the last two on day 6. Additional samples of the same size were also prepared by immersion in approximately 80 mL for 40 hours. 【0319】 After removing each sample from the ribose solution, it was washed with running water and then placed in a water bath for two days to wash away any unreacted ribose with PBS and sodium azide. The water was changed periodically (once or twice daily) to aid the washing process. The samples were then partially dried and frozen for further processing. 【0320】 Staining methods for ribose-bridged scaffolding: Two common methods are used to stain bridged scaffolding. One can be described as meta-staining, where MB and GV are added to the bridged solution. In this method, one 4 × 8 cm 2 The scaffold pieces were immersed for 24 hours in a solution consisting of 98 mL of standard / stock ribose solution (same as above) and 1 mL of each dye (MB / GV) stock solution, where the stock solution is 0.1% by weight, resulting in a MB / GV concentration of 0.002% in the solution. After the combined crosslinking / staining process, the samples were washed first with tap water and then left in water for 2 days, in the same manner as described above for crosslinking, to produce sample 1610 as shown in Figure 16. Sample 1610 in Figure 16 is a meta-stained, ribose-crosslinked, and stained scaffold that was left in a "meta" solution for 24 hours. 【0321】 According to the second method, the post-staining uses the same conditions as previously discussed for the "standard staining process," namely, after the scaffold has undergone the cross-linking and washing process, the sample is stained for 3 hours in a 0.002 wt% solution of MB / GV in PBS, for a total area of ​​4 × 4 cm. 2The scaffold pieces were stained in 100 mL of solution. This can be done using pre-crosslinked scaffolds, regardless of the crosslinking time, for example, 24 hours, 40 hours, 5 days, or 6 days. Figure 17 shows post-stained, ribose-crosslinked scaffold 1710, which was left for 40 hours after 3 hours of standard dye treatment in 0.002% MB / GVPBS solution. 【0322】 Other embodiments and exemplary methods may include modifications from the ribose crosslinking example described above. The meta-staining process can be modified in at least two ways. A first modification may involve increasing or decreasing the time in the solution. A second modification may involve changing the concentration of either the dye or ribose in the solution. Since dye absorption occurs relatively slowly over time and is directly related to the dye concentration in the solution, for example, if meta-staining is 48 hours, and the color concentration in the scaffold needs to be the same as in the 24-hour process described above, it may be productive to decrease the concentrations of MB and GV in the solution. In fact, any combination of time and concentration is possible (within reasonable limits) to obtain specific results. 【0323】 Regarding the post-staining described above, the scaffold cross-linking time can be changed. If changes in the dye concentration within the scaffold are enabled, the dye concentration and / or time in the dye solution can also be changed. 【0324】 Example 2 - Genipine Crosslinking In the second embodiment, as described above, genipine is used as a crosslinking agent, and an exemplary procedure for this is described here. 【0325】 A genipine crosslinked solution is prepared. In this example, a 0.3% (w / v) solution of genipine in PBS is prepared, and 200 mL of the solution is prepared by dissolving 0.60 g of genipine in 200 mL of a pre-made PBS solution (9.55 g of pre-made PBS powder / 1 L). The solution is stirred until no solid particles remain. 【0326】 In this example, Kerecis® fish skin-derived cell scaffold products were again used as a skin substitute. Generally, any size and / or number of scaffolds, including even granulated scaffold materials, could be processed. In this example, a culture plate with 15 mL wells was used. 2 × 2 cm 2 Place the scaffold / collagen pieces in each well and add the genipin solution. Each well was completely filled with a total of 15 mL of solution. Then, the plate was sealed with a lid and immersed in a 37°C water bath for 24 hours. Note that while the temperature is constant at 37°C, any other heat source may be present, and evaporation can be limited by sealing the container or re-condensing the solution. After 24 hours in the solution, the scaffold was washed with water and frozen. In this example, crosslinking with genipin caused the scaffold material to curl up, and the scaffold changed to a bluish-black color. There was also a clear difference in the rigidity of the samples. 【0327】 Similar to ribose crosslinking and staining, the two methods investigated in this example are staining during and after crosslinking, i.e., meta-staining and post-staining. In this example, six wells were used, four of which contained only 15 mL of 0.3% genipine solution, and two contained MB and GV (meta-staining). The meta-staining solution was prepared by adding 150 μL (0.1 wt%) of each dye stock solution to the well, followed by the addition of 14.7 mL of genipine solution. Except for the addition of MB / GV, all six wells were identical and underwent the same treatment during the crosslinking process. 【0328】 The post-staining procedure for genipin-crosslinked scaffolds is the same as for ribose-crosslinked scaffolds. Immerse the sample in 0.002% MB / GV, PBS solution for 3 hours and stain 2 × 2 cm samples. 2 Use 25 mL of solution for each. Here, use 50 mL of solution to produce two 2 × 2 cm samples, as shown in Figure 18. 2The scaffold pieces were stained. Figure 18 shows sample 1810, a post-stained genipin scaffold stained in 0.002 wt% MB / GV PBS solution for 3 hours. After staining, the samples were washed, partially dried, and frozen. 【0329】 Other embodiments and exemplary methods may include modifications from the genipin crosslinking example described above. Modifications that may be made to the staining process of the genipin crosslinked scaffold sample are essentially the same as those in the ribose method described above; that is, the time and concentration of the dye can be changed regardless of the process (meta or post-staining). 【0330】 Figures 19A and 19B show a comparison of the improvement in color retention by chemical crosslinking. Figure 19A shows a comparison of fragments 19-C, 19-B, and 19-A in dishes 1930, 1920, and 1910, respectively. Each of the samples from which fragments 19-C, 19-B, and 19-A were taken was Kerecis® fish skin-derived cell scaffold product left in 0.002% MB / GV, PBS solution for 3 hours. The sample from which fragment 19-C was taken was also crosslinked with 0.3% genipin solution according to Example 2 above. The sample from which fragment 19-B was taken was also crosslinked with ribose solution according to Example 1 above. The sample from which fragment 19-A was taken was not crosslinked, but was simply colored in 0.002% MB / GV, PBS solution for 3 hours. 【0331】 For comparison, Figure 19A shows the respective fragments 19-C, 19-B, and 19-A in stained dishes 1930, 1920, and 1910, as well as samples for 19-C and 19-B. Subsequently, a pH 8 bicarbonate solution was added to each of dishes 1930, 1920, and 1910 in equal volumes and concentrations. Fragments 19-C, 19-B, and 19-A were stored in dishes 1930, 1920, and 1910, respectively, at a temperature of 37°C for 48 hours, resulting in the same fragments 19-C, 19-B, and 19-A after 48 hours, as shown in Figure 19B. 【0332】 As can be seen, the staining fastness of fragments 19-C and 19-B, which were crosslinked with genipin (19-C) and ribose (19-B), respectively, was significantly improved compared to 19-A, which was similarly stained but not crosslinked. The crosslinked pieces 19-C and 19-B clearly demonstrate that their staining is faster and better maintained. 【0333】 Furthermore, it is worth noting that, in both cases with ribose and genipin, the concentration and time for the crosslinking process can also be modified. This affects the final color for both procedures in the case of meta-staining, but has a greater effect in the case of genipin, as the color obtained directly from crosslinking as a result when using the methods described above is very dark and concentrated. If the time, concentration, or temperature in the solution is reduced, as shown in several studies, the result is less crosslinking and a lighter color. 【0334】 Crosslinking by irradiation In another embodiment, the skin substitute is crosslinked by irradiating the skin substitute material with electromagnetic radiation. In the first example, the skin substitute, such as scaffolding material, is irradiated with ultraviolet (UV) radiation. 【0335】 UV crosslinking example According to the UV-based example, a 0.1% stock solution of methylene blue (MB) was prepared by adding 200 mg of MB to 200 mL of sterile water and stirring until the color dissolved. A PBS solution was also prepared by mixing 1 liter of liquid 10×PBS with 8.8 liters of tap water and stirring. 【0336】 Kerecis™ fish skin-derived cell scaffold products were again used as a skin substitute. Generally, any size and / or number of scaffolds can be processed, including granulated scaffold material, which can have a diameter as small as 1 mm. In this example, the fragments used included 14 fish skin pieces cut to 4 × 8 cm and 2 uncut fish skins. All fish skins were pre-scraped to remove muscle tissue, scales, and fascia. 【0337】 A portion of the PBS and stock solution were placed in a large container and stirred until uniform. The volume of each solution was 8.8 L for the PBS stock and 200 mL for the stock color. 【0338】 After preparing the colored solution, the fish skin was added to the solution and stirred to ensure that the fish skin did not stick together. 【0339】 The fish skin was left in a colored solution for 3.5 hours, stirring every hour. A UV cabinet was set up, and the fish skin was placed on a tray. 【0340】 The UV radiation source was 15W UV light (254nm), which was screwed into the inside of the cabinet so that the tray could be placed under the light during the radiation process. The inside of the cabinet was covered with aluminum foil and the light on the wall was again directed towards the fish skin. The tray was placed approximately 12cm away from the light. In this embodiment, UV light, which is almost monochromatic UV radiation, was selected, but in other embodiments, other UV sources of various wattages and wavelengths may be used, either as monochromatic or polychromatic UV radiation, with the UV radiation having wavelengths in the range of about 10nm to about 400nm. 【0341】 Therefore, we obtained samples including: Sample A, removed from an MB-based coloring solution, placed in a PBS solution (colorless), and exposed to UV radiation; Sample B, placed in an MB coloring solution and exposed to UV radiation while in the MB coloring solution; and Sample C, placed in a coloring solution but not exposed to UV radiation. In other words, the skin of Sample A can be considered similar to post-staining crosslinking, as it was stained in an MB-based coloring solution and then crosslinked by UV radiation only while in the PBS solution. By comparison, the skin of Sample B can be considered similar to meta-staining, as it remained in an MB-based coloring solution while being crosslinked by UV radiation. The skin of Sample C can also be considered a control, as it was stained in an MB-based coloring solution but not exposed to UV radiation during or after staining. However, as a control, the skin of Sample C was kept in a dark area of ​​a UV cabinet, but not exposed to UV radiation. In this way, the skin of sample C was treated under similar temperature, flipping, and time conditions for comparison with the cross-linked skins of samples A and B. 【0342】 The skin was left in each of the liquid solutions for 6 hours. Because the skin was floating, it was turned upside down to ensure that both sides were evenly exposed to UV light. This process was also performed on skin in a tray that had not been exposed to UV light (in the dark), and the same process was followed as with the skin under UV light. When turning the skin over, the UV light was turned off for approximately 5-10 minutes. 【0343】 The temperature of the liquid solution was measured when the skin was turned inside out. Since the maximum temperature after 5 hours was less than 25°C, it was concluded that the UV light did not heat the solution and skin to the extent that a cooling system would be necessary. 【0344】 After the radiation process, the skin samples were collected from the tray and transferred to three separate bags, one sample each for sample A, sample B, and sample C. The skin samples were rinsed with cold water for several minutes, placed on a steel plate, and then inserted into a freeze-dryer. The skin samples were left in the freeze-dryer overnight. 【0345】 The skin was collected in three separate bags, and each fragment was sealed in a Tyvek bag. 【0346】 Some fragments of various samples were sterilized using ethylene oxide. 【0347】 Sample A: Sample A skin was colored in MB color solution, then removed, rinsed, and placed in PBS solution under UV light. The PBS solution was initially colorless, but at the end of UV radiation, it was clear that color from the skin from the previous coloring had leaked out and colored the PBS solution. The final color of the skin was a lighter blue, and even slightly greenish, compared to other prototypes which were a darker blue. 【0348】 Samples B and C: When comparing fragments 20-B of Sample B and 20-C of Sample C, as seen in Figure 20A, once the cross-linking of Sample B's skin was complete, there was no visible difference between the colored skin of Sample B (Sample B) and the skin of Sample C (Sample C) that had been placed under UV light while in the MB-based color solution, compared to the skin of Sample C that had been exposed to UV light. For comparison, fragment 20-A of Sample A is also provided in Figure 20A. The skin of Samples B and C had very similar colors, and there was no visible difference in the texture or feel of the fish skin. 【0349】 No liquid solution samples were collected for color quantification. The prototype was created solely for testing UV radiation and its effect on fish skin. 【0350】 Other methods for measuring the amount of color in the skin may be used later, such as by breaking down a portion of fish skin with enzymes and measuring the amount of color in the resulting solution. 【0351】 To compare the color fastness of the crosslinked samples, color bleeding was performed on fragments of each sample by leaving them in basic and acid (acid / base) solutions at 37°C. Furthermore, the samples were compared to other prototypes that had been previously colored with different mordants and while gradually changing the pH of the solution. The results showed that the UV-crosslinked samples maintained their color longer than many of the other prototypes. 【0352】 Figures 20A–20D show a comparison of improvements in color retention due to chemical crosslinking by UV irradiation. Figure 20A shows a comparison of fragments taken from samples C, B, and A, including a fragment of sample C labeled 20-C and placed in dish 2030; a fragment of sample B labeled 20-B and placed in dish 2020; and a fragment of sample A labeled 20-A and placed in dish 2010. Figure 20B shows fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after immersion in the same concentration acid / base solution for 24 hours. Figure 20C shows fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after immersion in the same concentration acid / base solution for 48 hours. Figure 20CD also shows fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after immersion in acid / base solution of the same concentration for 72 hours. As can be seen, the stain fastness of fragments 20-B and 20-A, exposed to UV radiation, was significantly improved compared to fragment 20-C, which was similarly colored but not exposed to UV radiation. This is particularly true at 48 and 72 hours, respectively, in the acid / base solution, as seen in Figures 20C and 20D. At both 48 and 72 hours, fragments 20-B and 20-C still retained some color, but after 72 hours, the uncrosslinked fragment 20-C had become almost white or returned to its original color. A comparison of fragments 20-B and 20-A at both 48 and 72 hours suggests that the meta-staining of sample B, where the fish skin was stained with UV radiation while in an MB-based color solution, appears to result in slightly more colorfast fish skin upon exposure to an acid / base solution. Specifically, the color of fragment 20-B appeared slightly darker than that of fragment 20-A after immersion in acid / base solution for both 48 and 72 hours. 【0353】 Other embodiments and exemplary methods may include, but are not limited to, changes in the colorant, the intensity of UV radiation, the wavelength or wavelength range of UV radiation, the staining time, the staining density, and the time of exposure to UV radiation. 【0354】 Based on these examples and the embodiments described, the inventors have found and shown that the properties of colored skin substitutes, such as colored scaffolding materials, can be improved, including increasing the fastness of the colorants used to color the skin substitutes, improving the mechanical material properties of the skin substitutes, increasing the resistance of the skin substitutes to enzymatic and chemical degradation, and extending the lifespan of the colorants added to the skin substitutes under biological conditions such as treated wounds. 【0355】 Possible combinations of embodiments and features This disclosure provides various examples, embodiments, and features, but it should be understood that they are combinable with other examples, embodiments, or features described herein unless expressly stated or mutually exclusive. 【0356】 In addition to the above, further embodiments and examples include: 【0357】 1. A tissue regenerative wound treatment comprising a skin substitute and a coloring agent added to the skin substitute, wherein the coloring agent is a biocompatible coloring agent that decomposes when attacked by proteases within the treated wound. 【0358】 2. Tissue regenerative wound treatment by any of the above 1 or 3-14 below, or a combination thereof, wherein the skin substitute is a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute. 【0359】 3. Tissue regeneration wound treatment according to any of the above 1-2 or 4-14 or a combination thereof, wherein the skin substitute is an autologous skin graft, an allogeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft. 【0360】 4. Tissue regeneration wound treatment according to any of the above 1-3 or 5-14 or a combination thereof, wherein the skin substitute includes scaffolding material. 【0361】 5. Tissue regenerative wound treatment by any of the above 1-4 or 6-14 or a combination thereof, wherein the skin substitute includes a scaffold material containing an extracellular matrix product. 【0362】 6. Tissue regenerative wound treatment by any or a combination of the above items 1-5 or 7-14 below, wherein the extracellular matrix product is in the form of particles, sheets, or mesh. 【0363】 7. Tissue regeneration wound treatment according to any one or a combination of the above 1-6 or the following 8-14, wherein the skin substitute is a scaffold material containing intact decellularized fish skin, and the intact decellularized fish skin contains extracellular matrix material. 【0364】 8. A tissue regenerative wound treatment according to any one or a combination of items 1-7 above or items 9-14 below, wherein the wound treatment is cross-linked before, after, or between the addition of a coloring agent to the skin substitute. 【0365】 9. Tissue regeneration wound treatment according to any one or a combination of the above 1-8 or the following 10-14, wherein the coloring agent comprises a thiazine dye, a triarylmethane dye, or a combination of a thiazine dye and a triarylmethane dye. 【0366】 10. Tissue regeneration wound treatment according to any one or a combination of the above 1-9 or the following 11-14, wherein the coloring agent includes methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV). 【0367】 11. Tissue regeneration wound treatment according to any one or a combination of items 1-10 above or items 12-14 below, wherein the skin substitute is freeze-dried and a colorant is added to the skin substitute before freeze-drying or refrozen freezing of the skin substitute. 【0368】 12. Tissue regeneration wound treatment according to any one or a combination of the above 1-11 or the following 13-14, wherein the coloring agent is added to the skin substitute by staining the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the coloring agent in deionized water or phosphate-buffered saline. 【0369】 13. Tissue regeneration wound treatment by any one or a combination of the above 1 to 12 or the following 14, characterized in that the coloring agent has one or more properties among those of an antibiotic, preservative, antibacterial agent, antiviral agent, antifungal agent, antiparasitic agent, anti-inflammatory agent, or antioxidant agent. 【0370】 14. Tissue regeneration wound treatment using any or a combination of the above 1-13, wherein the coloring agent does not cause permanent discoloration of the wound upon healing. 【0371】 15. A wound treatment method comprising the steps of: providing a tissue regenerative wound treatment according to any one or a combination of the above 1 to 14; applying the tissue regenerative wound treatment to a wound bed; and determining whether the skin substitute is being degraded by protease attack within the wound by determining a change in the color of a coloring agent. 【0372】 16. A method for producing a tissue regenerating wound treatment, the method comprising the steps of providing a skin substitute and adding a colorant to the skin substitute, wherein the colorant is a biocompatible colorant that degrades when attacked by proteases within the treated wound. 【0373】 17. A method according to any one or a combination of 16 or 18-20 below, wherein the skin substitute is a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute, and / or the skin substitute is an autologous skin graft, an allogeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft, and / or the skin substitute comprises a scaffold material, and / or the skin substitute comprises a scaffold material comprising an extracellular matrix product. 【0374】 18. A method according to any one of the above 16-17 or the following 19-20 or a combination thereof, wherein the skin substitute is a scaffold material comprising intact decellularized fish skin, and the intact decellularized fish skin comprises an extracellular matrix material. 【0375】 19. A method according to any one or a combination of the above 16-18 or the following 20, wherein the coloring agent includes methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV). 【0376】 20. A method according to any one or a combination of the above 16 to 19, wherein the coloring agent is added to the skin substitute by staining the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the coloring agent in deionized water or phosphate-buffered saline. 【0377】 A simplified list of defined terms To aid in understanding the scope and content of the claims described above and attached to this document, several selected terms are defined below directly. 【0378】 As used herein, the term “base material” may include any material known in the art that can act as a vehicle for a therapeutic agent, and in addition to or alternatively, allow and / or passively regulate moisture in and / or around a wound. 【0379】 The term "biocompatible polymer" refers to polymer materials that are not harmful to the human body. Biocompatible polymers include synthetic or natural polymer materials that do not release substances harmful to the human body and do not cause side effects such as skin irritation or any other adverse effects on the human body, even when in direct contact with wound sites. 【0380】 As used herein, the degree of “Echelon” refers to the location and / or type of medical treatment provided to military personnel. Echelon I refers to treatment by combat medics, in addition to self-help and comrade-assisted treatment performed away from the battlefield or Echelon II personnel offices / facilities. Echelon II refers to advanced trauma care performed by physicians, physician's assistants, or other qualified medical personnel, and Echelon II care is often provided in field hospitals. Echelon III refers to care provided at the corps level, typically including reconstructive and curative surgeries to save life, limbs, and vision, and this care may be provided in field hospitals equipped with the necessary facilities. Echelon IV refers to complex surgeries and extended recovery periods (e.g., more than two weeks), and is generally provided in local permanent hospitals. Echelon V refers to injuries and / or procedures requiring extensive rehabilitation and recovery care, and Echelon V treatment is provided in permanent hospitals in the continental United States. The aforementioned echelon system is particularly relevant to military and intervention scenarios, but may be applied, as appropriate, to intervention locations and / or types of intervention scenarios in civilian and / or local law enforcement scenarios. 【0381】 The term “wound” as used herein is generally intended to encompass tissue damage. Therefore, the term “wound” includes injuries resulting in cuts, lacerations, and / or destruction of the skin, such as ruptures, abrasions, incisions, punctures, detachments, or other similar injuries. Wounds can be described by their size, shape, or scale. For example, a paper cut is an example of a relatively small, straight incision, while a concussive blast, resulting in a large laceration covering one or more body parts, is an example of a larger, relatively large wound. However, each of the aforementioned examples falls within the scope of the term “wound” as used herein. 【0382】 The term “wound” also includes damage to underlying tissues, such as those caused by trauma. Therefore, the term “wound” is intended to encompass combinations of multiple different wounds. For example, a traumatic cut from an explosion may generally be called a wound, even if it is a collection of various lacerations, abrasions, delaminations, and punctures. Furthermore, any damage to underlying tissues resulting from an explosion, as described above, may be further encompassed within the scope of this understanding of wounds. The term “wound” is also intended to encompass tissue injuries caused by burns (e.g., thermal and / or chemical burns). Furthermore, the term “wound” is also intended to encompass injuries resulting from other causes, such as diabetic foot ulcers, venous leg ulcers, surgical procedures, pressure ulcers, and others. 【0383】 As used herein, “traumatic wound” refers to any wound resulting from a physical injury that damages both the skin and underlying tissue. A gunshot wound is one non-limiting example of a traumatic wound, as it causes puncture (i.e., destruction) of the skin and rupture or otherwise damage to the underlying tissue. Another non-limiting example is a concussion or explosion, which generally result in a traumatic wound (one or more). Many, though not all, of wounds sustained during wartime can be described as traumatic wounds due to the nature of the war and war-related injury. “Traumatic wounds” may include hemorrhagic wounds, wounds exposing bone and / or tendons, severe burns, deep tissue wounds (e.g., asymmetric deep tissue wounds), and / or wounds of large surface areas. 【0384】 Omega3 Wound is approved by the Food and Drug Administration (FDA) for use in wound management, including chronic wounds and burn wounds, and soft tissue repair. Unlike other animal-derived products, fish skin requires gentle processing to preserve its structure and bioactive components, as there is no risk of disease transmission to humans. Omega3 Wound has been demonstrated to have advantages over porcine small intestine-derived scaffolds in terms of faster wound closure and rapid healing time. Fish skin grafts have been used for numerous chronic and acute wounds of various etiologies, demonstrating robust safety and efficacy. Given the complex and hostile environment of war, a comprehensive approach combining advanced wound care techniques and infection prevention measures should be taken. It is crucial that new technologies consider and address the needs of soldiers and healthcare workers. 【0385】 Various modifications and / or alterations to the features of the invention illustrated herein, and additional applications of the principles illustrated herein, which would be conceivable to a person skilled in the art and familiar with the relevant technology, may be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims and should be considered within the scope of this disclosure. Accordingly, although various aspects and embodiments are disclosed herein, other aspects and embodiments are also contemplated. Many methods and components similar to or equivalent to those described herein may be used to carry out embodiments of this disclosure, but only specific components and methods are described herein. 【0386】 Furthermore, it is understood that systems, devices, products, kits, methods, and / or processes according to specific embodiments of the Disclosure may include, incorporate, or otherwise possess characteristics, features (e.g., components, members, elements, parts, and / or sections) described in other embodiments disclosed and / or described in the Specification. Accordingly, various features of a particular embodiment may be compatible with, combined with, included in, and / or incorporated into other embodiments of the Disclosure. Therefore, the disclosure of certain features relating to a particular embodiment of the Disclosure should not be construed as limiting the application or inclusion of such features to a particular embodiment. Rather, it is understood that other embodiments may also include such features, members, elements, parts, and / or sections without necessarily departing from the scope of the Disclosure. 【0387】 Furthermore, unless otherwise stated, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. Moreover, various well-known embodiments, such as exemplary systems, methods, and apparatus, are not described in particular detail herein to avoid obscuring the embodiments of the exemplary models. However, such embodiments are also considered herein. 【0388】 It should be understood that not all objectives or benefits are necessarily achieved under the embodiments of this disclosure. Those skilled in the art will recognize that exoskeletons and methods for making exoskeletons may be embodied or performed in a manner that achieves or optimizes one benefit or group of benefits as taught herein, without achieving any other objectives or benefits as taught or suggested herein. 【0389】 Those skilled in the art will recognize some of the interchangeability of the various disclosed features. Those skilled in the art will also be able to mix and adapt other known equivalents for each feature to construct an exoskeleton and utilize methods for making an exoskeleton based on the principles of this disclosure, in addition to the modifications described herein. 【0390】 While this disclosure describes specific exemplary embodiments and examples of passive lumbar exoskeletons, it will be understood by those skilled in the art that this disclosure extends beyond the specifically disclosed embodiments of passive lumbar exoskeletons to other alternative embodiments and / or uses of this disclosure and obvious modifications and equivalents thereof. This disclosure is not to be limited by the embodiments disclosed above and is intended to be extended to other uses that may utilize the features described herein.

Claims

[Claim 1] Skin substitutes; and A colorant added to the aforementioned skin substitute, which is biocompatible and decomposes upon protease attack, enzymatic degradation, chemical degradation, or hydrolysis within the treated wound. A tissue regeneration wound treatment composition comprising, A tissue regeneration wound treatment composition wherein the skin substitute comprises a scaffolding material, and the scaffolding material comprises intact decellularized fish skin containing extracellular matrix material for infiltration and inward growth by host cells. [Claim 2] The tissue regeneration wound treatment composition according to claim 1, wherein the skin substitute further comprises a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute. [Claim 3] The tissue regeneration wound treatment composition according to claim 1 or 2, wherein the skin substitute further comprises an autologous skin graft, a syngeneic skin graft, an allogeneic skin graft, a heterogeneic skin graft, or a synthetic skin graft. [Claim 4] The tissue regeneration wound treatment composition according to any one of claims 1 to 3, wherein the host cells include endothelial cells and / or epithelial cells and / or fibroblasts, thereby making the scaffold material suitable for infiltration and inward growth by the endothelial cells and / or epithelial cells and / or fibroblasts of the host cells. [Claim 5] The tissue regeneration wound treatment composition according to any one of claims 1 to 4, wherein the extracellular matrix material is in the form of particles, sheets, or a mesh. [Claim 6] The tissue regeneration wound treatment composition according to any one of claims 1 to 5, wherein the tissue regeneration wound treatment composition is crosslinked. [Claim 7] The tissue regeneration wound treatment composition according to any one of claims 1 to 6, wherein the coloring agent comprises a thiazine dye, a triarylmethane dye, or a combination of a thiazine dye and a triarylmethane dye. [Claim 8] The tissue regeneration wound treatment composition according to any one of claims 1 to 7, wherein the coloring agent comprises methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV). [Claim 9] The tissue regeneration wound treatment composition according to any one of claims 1 to 8, wherein the skin substitute and the coloring agent added to the skin substitute are in a freeze-dried form. [Claim 10] The tissue regeneration wound treatment composition according to any one of claims 1 to 9, characterized in that the coloring agent has one or more properties among those of an antibiotic, preservative, antibacterial agent, antiviral agent, antifungal agent, antiparasitic agent, anti-inflammatory agent, or antioxidant agent. [Claim 11] The tissue regeneration wound treatment composition according to any one of claims 1 to 10, wherein the coloring agent does not cause permanent discoloration of the wound during healing. [Claim 12] A method for determining the breakdown of a skin substitute, A step of detecting a change in the color of the coloring agent of the tissue regeneration wound treatment composition according to claims 1 to 11 applied to a wound bed, A step in which, when the aforementioned change in color is detected, it is determined that the skin substitute is being broken down in the wound by protease attack, enzymatic degradation, chemical degradation, or hydrolysis. A method that includes this. [Claim 13] A method for producing a tissue regeneration wound treatment composition, wherein the method is The process of providing a skin substitute, A step of adding a coloring agent to the skin substitute, wherein the coloring agent is a biocompatible coloring agent that decomposes when subjected to protease attack, enzymatic degradation, chemical degradation, or hydrolysis within the treated wound. A method comprising, wherein the skin substitute comprises a scaffolding material, and the scaffolding material comprises intact decellularized fish skin comprising extracellular matrix material for infiltration and inward growth by host cells. [Claim 14] The skin substitute further comprises a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute, and / or The method according to claim 13, wherein the skin substitute further comprises an autologous skin graft, a syngeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft. [Claim 15] The scaffolding material includes intact decellularized fish skin, The method according to claim 13 or 14, wherein the intact decellularized fish skin comprises an extracellular matrix material. [Claim 16] The method according to any one of claims 13 to 15, wherein the coloring agent includes methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV). [Claim 17] The method according to any one of claims 13 to 16, wherein the coloring agent is added to the skin substitute by staining the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the coloring agent in deionized water or phosphate-buffered saline. [Claim 18] The method according to any one of claims 13 to 17, wherein the tissue regeneration wound treatment composition is crosslinked before, after, or simultaneously with the addition of the coloring agent to the skin substitute. [Claim 19] The method according to any one of claims 13 to 18, wherein the skin substitute is freeze-dried, and the colorant is added to the skin substitute before freeze-drying or refrozen-drying the skin substitute. [Claim 20] The tissue regeneration wound treatment composition according to any one of claims 1 to 11, wherein the coloring agent is a biocompatible coloring agent that decomposes when attacked by a protease.

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