A pigmented biological wound treatment that provides healing progress monitoring

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 relates generally to wound treatments and methods for stabilizing, protecting, and / or healing damaged tissue, and more particularly to wound treatments and methods that indicate whether ingrowth of a wound treatment into a skin substitute has occurred. [Background technology]

[0002] Healthy skin serves several different functions, including protecting the underlying tissues from abrasion, microorganisms, water loss, and damage caused by ultraviolet light. The nervous system of healthy, normal skin also provides the tactile senses of touch, pressure, and vibration, the thermal senses of hot and cold, and the sensation of pain. Body thermoregulation relies on the skin's ability to sweat and control blood flow to the skin to increase or decrease heat loss. Healthy skin contains three distinct 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 flattened, keratinized, dead keratinocytes that form a barrier against water loss and microbial invasion. The dead keratinocytes originate from living keratinocytes in the basal layer above the dermis and are responsible for re-epithelialization of the skin. The epidermis does not contain nerves or blood vessels and receives water and nutrients via diffusion from the dermis. The dermis, which lies beneath the epidermis, is composed mostly of collagen fibers produced by fibroblasts and some elastic fibers, which together with water and large proteoglycan molecules constitute 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, which form a thick layer of adipose tissue.

[0003] A wound may be considered a disruption in the structural and functional integrity of the skin. Thus, a "wound" may include an injury that causes a cut, tear, and / or disruption of the skin, such as, for example, a laceration, abrasion, incision, puncture, abrasion, burn, or other similar injury.

[0004] After hemostasis, which often occurs after a wound event, wounds undergo the main stages of healing: inflammation, proliferation, and remodeling. Chronic wounds can be considered as wounds that have failed to go through the normal healing process in an orderly and timely manner. Chronic wounds often remain in the inflammatory stage.

[0005] In many cases, in cases of serious wounds, such as wounds that extend over a wide area or deep wounds, large or severe burns, or in cases of chronic wounds, skin substitutes are often used to aid the wound healing process and to more quickly restore at least some of the functions of healthy skin described above. Skin substitutes can be broadly considered as a group of elements or materials that allow temporary or permanent closure of a wound. Skin substitutes can be generally classified as biological skin substitutes, synthetic skin substitutes, or hybrid skin substitutes that include biological and synthetic skin substitutes.

[0006] Biological skin substitutes often have a more intact extracellular matrix structure, whereas synthetic skin substitutes can be synthesized on demand and tailored to specific purposes. Biological and synthetic skin substitutes each have advantages and disadvantages. Biological skin substitutes may allow for a more natural construction of new dermis and may allow for superior re-epithelialization properties due to the presence of a basement membrane. Synthetic skin substitutes may be chemically synthesized, offering the advantage of increased control over the scaffold composition. Synthetic skin substitutes include, for example, synthetic collagen or protein-based matrices, or synthetic biolayers that include collagen or protein-based components combined with silicone components. Hybrid skin substitutes may 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 goal of using a skin substitute is to provide effective, timely, scar-free wound healing while restoring as much as possible the function of the skin prior to the wounding event.

[0008] Examples of commercially available synthetic skin substitutes include Biobrane®, Dermagraft®, Integra®, Apligraf®, MatriDerm®, OrCel®, Hyalomatrix®, and Renoskin®.

[0009] US Patent Publication No. 2003 / 0059460 discloses a hybrid polymer skin substitute material containing synthetic and natural polymers that can be used for regenerating living tissue. The hybrid contains a crosslinked naturally occurring polymer and a biodegradable absorbable synthetic polymer. However, a series of complicated process steps must be performed to produce the hybrid material. 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 adequate moisture level on the wound. The products typically accumulate wound exudate and are changed periodically.

[0011] Biological skin substitutes may include skin grafts including, but 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 amniotic membrane grafts.

[0012] Additionally, in recent years, a new class of biological skin graft products has emerged that are intended to improve the wound microenvironment by providing shelter for proliferating cells. Typically, the new products are made from biological materials that contain intact collagen or reconstituted collagen. Examples include brands such as Oasis, Matristem, Integra, and Puracol. These products are often referred to by clinicians as matrix products. The matrix product is inserted into the wound where it attracts cellular ingrowth. 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. Pat. No. 8,613,957, which was granted on December 24, 2013. The decellularized fish skin product described in U.S. Pat. No. 8,613,957 functions as a scaffolding material that provides an intact scaffold to support the ingrowth of endothelial and / or epithelial cells. Decellularized fish skin scaffold material is 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. Fish skin is structurally similar to human skin with three basic layers including the epidermis, dermis, and subcutis, and contains proteins, lipids, fatty acids, and other bioactive compounds that are homologous to human skin.

[0013] Other examples of biological skin substitutes include those described in U.S. Pat. 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, CN Patent No. 1068703 describes a process for preparing fish skin for dressing burns, which involves separating the fish skin from the body of the fish and placing the skin in a preservative solution of tincture of iodine, ethanol, borneol, zinc sulfadiazine, and hydrochloric acid in a sufficient amount to establish a pH value of 2.5 to 3. However, these products can be difficult to handle because the product in U.S. Pat. No. 6,541,023 is in the form of a gel and the product in CN Patent No. 1068703 is preserved in a 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 intestinal submucosa (OASIS® Wound Matrix (Healthpoint Ltd.)).

[0015] As mentioned above, during healing, wounds go through three main phases: inflammation, proliferation, and remodeling. During the inflammation phase, the body secretes proteases into the wound to remove damaged tissue and debris from the wound. In some cases, when a skin substitute such as an extracellular matrix is ​​inserted into the wound, the proteases attack the skin substitute and break it down as if it were damaged tissue or debris. In other cases, the skin substitute such as an extracellular matrix functions as intended with cellular ingrowth to provide shelter for the proliferating new cells.

[0016] Clinicians using matrix products typically inspect the wound 1-3 days after initial application of the matrix product to the wound bed. A significant challenge identified by the inventors is the inability of clinicians and medical personnel to easily and / or accurately distinguish between degraded skin substitutes that have become sloughed tissue and pus substitutes or skin substitutes such as matrix that are wet and permeated by cellular ingrowth. The inventors have discovered that distinguishing between skin substitutes and skin substitutes in degraded wounds that are healing properly, i.e., permeated by cellular ingrowth, in the case of matrix skin substitutes for example, is critical to efficient healing of wounds. If the added skin substitute material has degraded or part of it has degraded, it is necessary to remove the degraded skin substitute material and clean the wound to remove the sloughed tissue and pus that is often contained in the degraded skin substitute material. After cleaning and removing the sloughed 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 been penetrated by cellular ingrowth, as intended, the matrix is ​​left in place and monitoring of the matrix material continues until the wound has healed appropriately.

[0017] For example, when using a fish skin derived cell scaffold product to heal wounds (e.g., as described in U.S. Patent No. 8,613,957, granted December 24, 2013), the inventors have found that clinicians and care providers unconsciously err or otherwise struggle to distinguish between the wound healing scaffold and an infection. This may be due, at least in part, to the color and / or odor associated with the wound healing scaffold as it degrades and begins to incorporate into the surrounding tissue, which some interpret as an odor similar to that of infected tissue, as it may sometimes have a color similar to infected tissue (e.g., suppurating infection) and may be mildly aromatic.

[0018] Thus, the inventors have further determined that without an efficient and effective means of determining whether the skin substitute has been permeated by cellular ingrowth, unnecessary removal or replacement of dressings would be required to inspect the wound, exposure of the wound, and unnecessary reapplication of the created skin substitute, which would prevent proper healing of the wound.

[0019] In addition, infection is a major challenge in wound healing and management. For example, in the case of combat wounds, infection determines the morbidity and mortality of military personnel injured on the battlefield. Infection accounts for one-third of all casualties, prolonging treatment and increasing the risk of amputation. Due to the unique mechanism of injury and the harsh environment, combat wounds are prone to contamination, making them more difficult to treat. An early sign of infection is bacterial imbalance within the wound. Common pathogens found in wounds at early stages include both Gram-positive (G+) and Gram-negative (G-) strains. Once infection occurs, emergence of Gram-negative and multi-drug resistant (MDR) bacteria is observed. There is a strong need for effective and immediate interventions to reduce the risk of infection to benefit soldiers and act as force multipliers in the combat zone.

[0020] Thus, in addition to providing a means of determining whether the skin substitute is permeated by cellular ingrowth, the inventors have further identified the challenge of making the skin substitute itself less susceptible to infection. Summary of the Invention

[0021] To address the above problems, the inventors herein disclose an ingrowth-indicative wound treatment comprising a skin substitute and a colorant applied to the skin substitute, the colorant being a biocompatible colorant that degrades upon attack by proteases within the treated wound.

[0022] Further provided is a method of wound treatment comprising providing a tissue regeneration wound treatment composition, applying the tissue regeneration wound treatment composition to a wound bed, and determining whether a skin substitute has been degraded within the wound by protease attack by determining a change in color of the colorant. The tissue regeneration wound treatment comprises a skin substitute and a colorant applied to the skin substitute. The colorant is a biocompatible colorant that degrades upon protease attack within the treated wound.

[0023] A method of producing a tissue regeneration wound treatment is provided, the method comprising the steps of providing a skin substitute and adding a colorant to the skin substitute, the colorant being a biocompatible colorant that degrades upon attack by proteases within the treated wound.

[0024] According to 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, a syngeneic 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 that includes 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 comprising intact decellularized fish skin, the intact decellularized fish skin comprising 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 colorant includes methylene blue (MB), or 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 lyophilized, where the colorant is added to the skin substitute prior to lyophilization or re-lyophilization of the skin substitute.

[0033] According to one or more embodiments, the colorant is added to the skin substitute by dyeing the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the colorant in deionized water or phosphate buffered saline.

[0034] According to one or more embodiments, the colorant is characterized as having one or more of the following properties: antibiotic, antiseptic, antibacterial, antiviral, antifungal, antiparasitic, anti-inflammatory, or antioxidant.

[0035] According to one or more embodiments, the tissue regeneration wound treatment further comprises an additional active agent comprising one or more of an antibiotic, an antiseptic, an antibacterial agent, an antiviral agent, an antifungal agent, an antiparasitic agent, an anti-inflammatory agent, an antioxidant, a drug, a protein, a peptide, or a combination thereof.

[0036] According to one or more embodiments, the coloring agent does not cause permanent coloring of the wound as it heals.

[0037] As described herein, the tissue regeneration treatment and tissue regeneration treatment method disclosed herein provide wound healing progress monitoring and provide an accurate, efficient and effective means to distinguish between deteriorated applied skin substitutes applied to wounds and skin substitutes that have been successfully infiltrated by cellular ingrowth.This allows clinicians or medical personnel to easily distinguish whether they (1) need to clean the wound and remove the unsuccessfully applied skin substitutes with sloughed tissue and pus, or (2) leave the applied skin substitute in place, and a coloring agent is added to the skin substitute, which has the property of being degraded by protease attack in the treated wound.That is, the coloring agent is added to the skin substitute, for example, during the manufacturing stage, and the color of the coloring agent is characterized by being changed, removed, or degraded by one or more proteases in the treated wound.

[0038] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an indication of the scope of the claimed subject matter.

[0039] Additional features and advantages of the present disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the present disclosure. The features and advantages of the present disclosure may be realized and obtained by means of the instructions and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.

[0040] These and other inventive features, aspects, and advantages of the present disclosure will become better understood with reference to the following description, appended claims, and accompanying drawings. [Brief description of the drawings]

[0041] [Figure 1A] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 1B] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 1C] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 1D] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 1E] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 1F] 1 illustrates an embodiment of a skin substrate according to the present disclosure. [Figure 2A] 1 illustrates an embodiment of a skin substrate according to the present disclosure in the form of decellularized fish skin. [Figure 2B] 1 illustrates an embodiment of a skin substrate according to the present disclosure in the form of decellularized fish skin. [Figure 2C] 1 illustrates an embodiment of a skin substrate according to the present disclosure in the form of decellularized fish skin. [Figure 2D] 1 illustrates an embodiment of a skin substrate according to the present disclosure in the form of decellularized fish skin. [Figure 2E] 1 illustrates an embodiment of a skin substrate according to the present disclosure in the form of decellularized fish skin. [Diagram 3] 1 illustrates a pigmented skin substitute according to an embodiment of the present disclosure. [Figure 4A] 1 illustrates various pigmented skin substitutes according to embodiments of the present disclosure. [Figure 4B] 1 illustrates various pigmented skin substitutes according to embodiments of the present disclosure. [Figure 4C] 1 illustrates various pigmented skin substitutes according to embodiments of the present disclosure. [Diagram 5] 1 illustrates a pigmented skin substitute according to an embodiment of the present disclosure. [Figure 6A] 1 illustrates various mordanted and colored skin substitutes according to embodiments of the present disclosure. [Figure 6B] 1 illustrates various mordanted and colored skin substitutes according to embodiments of the present disclosure. [Figure 6C] 1 illustrates various mordanted and colored skin substitutes according to embodiments of the present disclosure. [Figure 6D] 1 illustrates various mordanted and colored skin substitutes according to embodiments of the present disclosure. [Figure 7] 1 illustrates various pigmented skin substitutes dyed under pH grading according to embodiments of the present disclosure. [Figure 8] 1 illustrates a pigmented skin substitute according to an embodiment of the present disclosure. [Figure 9A] 1 illustrates a pigmented skin substitute according to an embodiment of the present disclosure before and after exposure to collagenase. [Figure 9B] 1 illustrates a pigmented skin substitute according to an embodiment of the present disclosure before and after exposure to collagenase. [Figure 10A] 1 shows a patient's wound before and after treatment according to methods and embodiments of the present disclosure. [Figure 10B] 1 shows a patient's wound before and after treatment according to methods and embodiments of the present disclosure. [Figure 11A] 1 shows a patient's wound before and after treatment according to methods and embodiments of the present disclosure. [Figure 11B] 1 shows a patient's wound before and after treatment according to methods and embodiments of the present disclosure. [Figure 12A] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12B] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12C] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12D] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12E] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12F] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12G] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12H]1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12I] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12J] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12K] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12L] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 12M] 1A-1D show a patient's wound before, during, and after treatment according to methods and embodiments of the present disclosure. [Figure 13] 1 illustrates an exemplary method of treating a wound using a tissue regeneration wound treatment according to an embodiment of the present disclosure. [Figure 14A] 1 illustrates the results of a bacterial inhibition / reduction assay according to embodiments of the present disclosure. [Figure 14B] 1 illustrates the results of a bacterial inhibition / reduction assay according to embodiments of the present disclosure. [Figure 14C] 1 illustrates the results of a bacterial inhibition / reduction assay according to embodiments of the present disclosure. [Figure 15A] 1 illustrates a comparison of skin grafts according to an embodiment of the present disclosure. [Figure 15B] 1 illustrates a comparison of skin grafts according to an embodiment of the present disclosure. [Figure 15C] 1 illustrates a comparison of skin grafts according to an embodiment of the present disclosure. [Figure 16] 1 illustrates an embodiment of a crosslinked, dyed scaffold material. [Figure 17] 1 illustrates another embodiment of a crosslinked, dyed scaffold material. [Figure 18] 1 illustrates another embodiment of a crosslinked, dyed scaffold material. [Figure 19A] 1 shows a comparison of color fastness of embodiments of crosslinked and dyed scaffolding materials. [Figure 19B]1 shows a comparison of color fastness of embodiments of crosslinked and dyed scaffolding materials. [Figure 20A] 13 shows a comparison of the color fastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20B] 13 shows a comparison of the color fastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20C] 13 shows a comparison of the color fastness of other embodiments of crosslinked and dyed scaffolding materials. [Figure 20D] 13 shows a comparison of the color fastness 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 not intended to limit the scope but to provide an exemplary description. The drawings illustrate exemplary configurations of a wound treatment according to the present disclosure and its features and subcomponents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] A better understanding of various embodiments of the present disclosure may be had from the following description taken in conjunction with the accompanying drawings, in which like reference numerals refer to like elements and in which:

[0044] While the disclosure is susceptible to various modifications and alternative constructions, specific exemplary embodiments are shown in the drawings described below. It is to be understood, however, that there is no intention to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention of the present invention is to cover all modifications, alternative constructions, combinations, and equivalents falling within the spirit and scope of the present invention.

[0045] The references used are provided for convenience only and therefore do not define the scope of protection or embodiments.

[0046] Unless a term is expressly defined in this application to have a stated meaning, it is understood that there is no intention, either explicitly or implicitly, to limit the meaning of such term beyond its plain or ordinary meaning.

[0047] Any claim element that does not expressly recite a "means for" performing a particular function or "steps for" performing a particular function is not to be construed as a "means" or "steps" clause as specified in 35 U.S.C. § 112.

[0048] Skin substitute(s) As mentioned above, many different types of skin substitutes can be used to aid in the wound healing process and restore at least some of the functionality of healthy skin more quickly. Skin substitutes can be broadly considered as a group of elements or materials that allow for temporary or permanent closure of a wound. Skin substitutes can be generally classified as biological skin substitutes, synthetic skin substitutes, or hybrid skin substitutes that include biological and synthetic skin substitutes.

[0049] An example of such a skin substitute is shown in Figures 1A through 1F.

[0050] FIG. 1A shows an example of a skin substitute 100 for wound treatment according to an embodiment, comprising a first shredded decellularized fish skin particle 102. FIG. 1B shows an example of a skin substitute 110 for wound treatment according to an embodiment, comprising a second shredded decellularized fish skin particle 112. FIG. 1C shows an example of a skin substitute 120 for wound treatment according to an embodiment, comprising a third shredded decellularized fish skin particle 122. In the embodiment of FIG. 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. Pat. 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 generated from processed tilapia fish skin. Tilapia-based skin grafts can be provided in a variety of sizes, including large skin grafts 132, medium skin grafts 134, and small skin grafts 136. Figure 1E shows an example of a pig skin graft 140, including a non-meshed pig skin graft 142 and a meshed pig skin graft 144. A further example of a skin substitute is shown in Figure 1F, in this case a synthetic skin substitute 150, a bioengineered skin substitute formed from a bilayered tissue 152. In this non-limiting example, the bilayered tissue 152 of the dermis layer of the synthetic skin substitute 150 is a type I bovine collagen gel seeded with live human neonatal fibroblasts. The epidermis is neonatal keratinocytes. The cells actively secrete growth factors, cytokines, and extracellular matrix (ECM) proteins. Non-limiting examples of such synthetic skin substitutes include Apligraf™, which can be used to treat diabetic foot ulcers and venous leg ulcers.

[0052] 2A and 2B, an exemplary embodiment of a decellularized fish skin piece 200, 210 is described. An exemplary cross-section of a decellularized fish skin 200 made as described in U.S. Pat. No. 8,613,957 is illustrated in FIG. 2A with a size given by the size of a user's gloved hand 202. The size of the decellularized fish skin 200 is of course not limited and can be produced or provided or trimmed to fit the size and shape of the wound to be treated. Furthermore, although the decellularized fish skin shown is a non-meshed fish skin, a meshed decellularized fish skin may also be used.

[0053] It should be understood that the decellularized fish skin may be granulated, ground, or otherwise processed into various sizes and shapes. As shown in Fig. 2B, the multiple decellularized fish skin sheets 210 can be of a size and shape (e.g., rectangular) similar to the decellularized fish skin 200 of Fig. 2A, or can have a more uniform dimensional (e.g., square) shape, such as the decellularized fish skin sheet 220 illustrated in Fig. 2B.

[0054] The decellularized fish skin scaffold 210, 220 shown in Figures 2A and 2B is substantially rigid and inelastic in its freeze-dried form. The decellularized fish skin scaffold can be treated with one or more enzymes that act to increase its ductility and / or elasticity. In some embodiments, the enzymes act by cleaving interconnected extracellular matrix components without substantially affecting the healthy properties 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 significant portion of the extracellular matrix contents even if partially removed from its native three-dimensional structure.

[0055] In some embodiments, the enzyme treatment adversely affects the use of the modified decellularized fish skin as a scaffold material. However, it should be understood that, surprisingly, the loss of function as a scaffold material does not significantly affect the use of the 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 although this may adversely affect the use of the material as a scaffold for wound healing, the modified decellularized fish skin can still function as a wound preservation / stabilization material.

[0056] The decellularized fish skin scaffold can be ground to provide it in the form of particles. It should be understood that the size of the individual ground particles can vary depending on the type and / or method of grinding. For example, the decellularized fish skin particles can be created through a jet milling process designed to output particles below a specified size. In some embodiments, the decellularized fish skin is cut, chopped, or ground into particles, which can be done in a measured manner to create uniform particles, or it can be done roughly, which generates particles of various different sizes.

[0057] FIG 2C illustrates an exemplary depiction of large particles 232 of particulate or comminuted decellularized fish skin 230 resulting from grinding a sheet of decellularized fish skin scaffold material with a grinder, e.g., a cannabis grinder. FIG 2D illustrates an exemplary depiction of thread-like, cotton-like fibers 242 of particulate or comminuted decellularized fish skin 240 resulting from grinding a sheet of decellularized fish skin scaffold material with a grinder, according to an embodiment of the present disclosure. FIG 2E is an exemplary depiction of small, powder-like particles 252 of comminuted decellularized fish skin 250 resulting from grinding a sheet of decellularized fish skin scaffold material with a grinder, e.g., a cannabis grinder.

[0058] In an embodiment, the wound treatment is or comprises at least one particulate, particularly chopped, decellularized fish skin particle of a predetermined size. The particulate, i.e. chopped, decellularized fish skin particle is configured to provide a scaffolding material to support cell migration, attachment, proliferation, and differentiation to promote tissue repair and / or replacement, as described in U.S. Patent No. 8,613,957, filed October 6, 2010, and granted 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, the most abundant of which are collagens and other specialized proteins and proteoglycans. The scaffold material described herein is a mostly intact acellular scaffold of natural biological ECM components from fish skin. The scaffold can also include lipids that naturally occur from fish skin. The native three-dimensional structure, composition, and function of the dermal ECM are essentially unchanged, providing a scaffold that supports cell migration, attachment, proliferation, and differentiation, thus facilitating tissue repair and / or replacement.

[0060] The scaffold material according to the present invention is obtained from intact fish skin. Any kind of fish, including bony and cartilaginous fish, can be used as a source of fish skin. For example, the source can be round fish such as cod, haddock, and catfish, flatfish such as halibut, plaice, and flounder, salmonids such as salmon and trout, mackerel such as tuna, or small fish such as herring, anchovy, mackerel, and sardine. In certain embodiments, the fish skin is obtained from oily cold water fish and / or fish known to contain large amounts of omega-3 oil. Examples of fish high in omega-3 oil include salmon, sardine, tuna, herring, cod, sardine, mackerel, sable, smelt, white fish, hoki, and some types of trout.

[0061] Fish skin is removed from fish before processing. If the fish skin is from a species of fish that has scales, the scales of the fish skin must be removed so that most of the scales are removed or at least the hydroxyapatite is removed from the scales. The phrases "most of the scales are removed" or "substantially free of scales" mean that at least 95%, preferably at least 99%, more preferably 100% of the scales on the fish skin are removed. "Substantially free of scales" fish skin can also refer to fish skin from a fish species that does not have scales. The scales are removed using purely mechanical pressure (e.g., via a knife, shaking with an abrasive, water pressure, a special scale removal device that uses the same mechanical force as a knife, or other pressure devices such as grinding with ceramic or plastic) before any processing, or using mechanical pressure to wash off the scales after any chemical processing (e.g., decellularization). If the fish skin is first treated chemically and / or enzymatically (e.g., treatment with TRITON® X-100), the mechanical pressure generally needs to be gentle since it is susceptible to tearing after decellularization. The scales can be removed in more than one step, for example, partially removed prior to decellularization, followed by further removal during and / or after decellularization. Alternatively, the scales can be removed by chemical treatment alone.

[0062] After the scales are removed, the fish skin is optionally frozen prior to decellularization. The fish skin can be rapidly frozen by incubating the skin in liquid nitrogen or using other specialized freezing equipment that can freeze the skin below -70°C to preserve the collagen structure of the scaffold. Alternatively, the fish skin can be frozen in a conventional freezer typically found in aquaculture factories. The freezing process can help to dissolve or partially dissolve the cells that comprise the intact fish skin, facilitating the decellularization of the fish skin. If frozen, the fish skin can be thawed later for further processing.

[0063] The fish skin, whether frozen or not, can be washed with a buffer solution before further processing. For example, the fish skin can be washed 1-3 times with a buffer solution 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, phosphoramidon, and PMSF) to promote disinfection and stabilization of the fish skin. The buffer solution can be 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, 10.0 or more. 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 medium in which the fish skin can be stored for several days to several weeks or more. In certain embodiments, the fish skin is stored in the buffer at a temperature of about 4° C.

[0064] After freezing and / or washing and / or storage in buffer, the fish skin is treated with one or more decellularization solutions to remove cellular material, including antigenic substances, from the fish skin with minimal or no damage to the mechanical and structural integrity and biological activity of the naturally occurring extracellular matrix.

[0065] The term "extracellular matrix" or "ECM" as used herein refers to the non-cellular tissue material present within fish skin that provides structural support for 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 or reconstituted 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 or reconstituted from extracted, purified, or isolated ECM components (e.g., collagen).

[0066] As used herein, the terms "acellular", "decellularized", "decellularized fish skin" and the like refer to fish skin from which a significant amount of cellular and nucleic acid content has been removed, leaving behind a complex three-dimensional interstitial structure of ECM. In embodiments, "decellularized fish skin" may further include fish skin that contains omega-3 polyunsaturated fatty acids (PUFAs) in addition to the complex three-dimensional interstitial structure of ECM that does not contain a significant amount of cellular and nucleic acid content.

[0067] A "decellularization agent" is an agent that is effective in removing a substantial amount of cellular and nucleic acid content from the ECM. An ECM is "decellularized" or "substantially free" of cellular and nucleic acid content (i.e., a "substantial amount" has been removed) when at least 50% of the viable and nonviable nucleic acids and other cellular material have been removed from the ECM. In certain embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% of the viable and nonviable nucleic acids and cellular material are removed. Decellularization can be confirmed, for example, by testing the DNA content of the treated fish skin. Removal of nucleic acids from the 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 disrupts cell membranes and releases 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 freeze / thaw. Examples of chemical decellularization agents include ionic salts (e.g., sodium azide), bases, acids, detergents (e.g., non-ionic 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 DNA (e.g., poly-L-lysine, polyethylimine (PEI), and polyamine dodecylamine (PAMAM)). Non-ionic 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 zwitterionic detergents (e.g., CHAPS). Other suitable decellularization 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 decellularization agents include proteases, endonucleases, and exonucleases. Proteases include serine proteases (e.g., trypsin), threonine proteases, cysteine ​​proteases, aspartic acid proteases, metalloproteases (e.g., thermolysin), and glutamic acid proteases. 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, 10.0 or higher.In certain embodiments, the pH is between 7.0 and 9.0, such as between 7.5 and 8.5.

[0069] An example of a decellularization step is incubating fish skin in a solution containing 1M 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 ingredients (e.g., 0.02% sodium azide). The first decellularization solution is poured off, 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 ingredients (e.g., 0.02% sodium azide). In another example, fish skin is first treated with a decellularization solution containing a detergent (e.g., 0.5% TRITON® X-100) along with other ingredients (e.g., 0.02% EDTA, sodium azide, and / or deoxyformic acid) and then incubated in a second decellularization solution containing a detergent such as SDS.

[0070] The fish skin may or may not be cultured with shaking. The decellularization step(s) can be repeated, if desired, by pouring off the remaining decellularization solution, optionally washing the fish skin with a buffer (e.g., Hank's Balanced Salt Solution), and then subjecting the fish skin to another decellularization step again. Once a sufficient amount of cellular material has been removed, the decellularization solution can be removed (e.g., by aspiration or by gently pouring off the solution).

[0071] After decellularization, the fish skin can be optionally washed with water, buffer, and / or salt solution. Examples of suitable washing solutions include Dulbecco's Phosphate Buffered Saline (DPBS), Hank's Balanced Salt Solution (HBSS), Medium 199 (M199, SAFC Biosciences, Inc.), and / or L-glutamine. The washing step(s) 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, 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example 7.5 and 8.5.

[0072] To improve the appearance of the final product, the fish skin can be optionally bleached. Bleaching can be performed before, after, and / or simultaneously with decellularization. For example, one or more bleaching agents can be incorporated into one or more of the decellularization solutions and / or one or more buffers. Examples of bleaching agents include sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate. In certain embodiments, when a strong bleaching agent such as persulfate(s) is used, bleaching and decellularization can be combined in a single step that includes incubating the fish skin in a mixture of one or more bleaching agents, a thickening agent, and a peroxide source. For example, a dry bleaching mixture can be prepared (see, for example, "Bleaching Mixture" described in Example 5), followed by adding water, hydrogen peroxide, or a combination thereof to the dry mixture to form a bleaching solution that may also be sufficient for decellularization. Bleaching agents (e.g., sodium sulfite, hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate) should be about 40-60% w / w dry mix. A combination of EDTA and persulfate may be added to the mix 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 can be about 15-25% of the mixture, and the peroxide source can be sodium percarbonate and potassium percarbonate. Sodium phosphate perhydrate and sodium carbonate or magnesium metasilicate and silicate silica can also be used as peroxide sources. The dry mixture can also include, for example, 1-10% w / w 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 can optionally include thickening agents, 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 / decellularization solution and protect the 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, 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, for example 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. As with bleaching, digestion can be performed before, after, and / or simultaneously with decellularization. Suitable enzymes include proteases, such as serine proteases, threonine proteases, cysteine ​​proteases, aspartic acid proteases, metalloproteases, and glutamic acid proteases. In certain embodiments, the digestive enzyme is a serine protease, such as trypsin. The digestive enzyme can be an enzyme that functions in an alkaline environment to limit cross-linking within the ECM and soften 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, 10.0 or higher. In certain embodiments, the pH is between 7.0 and 9.0, such as between 7.5 and 8.5.

[0075] The decellularized fish skin can be optionally cryopreserved. Cryopreservation can involve immersing the fish skin in a cryoprotectant solution before freezing. The cryoprotectant solution generally includes a suitable buffer, one or more cryoprotectants, and optionally a solvent, such as an organic solvent combined with water to minimize swelling and shrinkage. Examples of cryoprotectants include sucrose, raffinose, dextran, trehalose, dimethylacetamide, methylsulfoxide, 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 crystals formed during the freezing process, the fish skin can be optionally frozen in a buffer solution that does not contain a cryoprotectant. Cryopreservation 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, 10.0 or higher, In certain embodiments, the pH is between 7.0 and 9.0, for example 7.5 and 8.5.

[0076] The decellularized fish skin can be packaged in a sterile container, such as a glass vial or pouch. In one embodiment, a TYVEK® pouch is used. For example, the fish skin can be incubated in a cryoprotectant solution, packaged in a TYVEK® pouch, and then placed in a freeze dryer to freeze at a rate compatible with the cryoprotectant.

[0077] The decellularized fish skin can be freeze-dried, i.e. frozen, under low temperature and vacuum conditions such that water is sequentially removed from each ice crystal phase without ice recrystallization. During freeze-drying, water is generally first removed by sublimation and then, if necessary, by desorption. Another method of removing excess water after processing and before sterilization is vacuum pressing.

[0078] In certain embodiments, the decellularized fish skin is sterilized before and / or after freezing. Sterilization methods are well known in the art. For example, the 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 by ozone, carbon dioxide, gaseous formaldehyde, or radiation (e.g., gamma radiation, X-rays, electron beam treatment, and subatomic particles).

[0079] As an alternative or in addition to aqueous freezing, freeze-drying, and / or vacuum pressing, the decellularized fish skin can be stored in a non-aqueous solution, such as alcohol.

[0080] The resulting product (scaffold material) is a sterile, collagen-based matrix with properties that may promote tissue regeneration, repair, and / or replacement (e.g., repair, regeneration, and / or growth of endogenous tissue). The term "scaffold material" in reference to fish skin refers to a material that includes fish skin that has been decellularized and optionally bleached, digested, freeze-dried, etc., as discussed above. The scaffold material can provide an intact scaffold for supporting endothelial and / or epithelial cells, can be incorporated by the host, is biocompatible, does not significantly calcify, and can be stored and transported at ambient temperature. The phrase "incorporated by the host" as used herein means that the cells and tissues of a patient being treated with the scaffold material can grow within the scaffold material, and the scaffold material is actually incorporated / absorbed into 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., is non-antigenic).

[0081] This may be measured by the ability of the material to pass biocompatibility tests set forth in the International Organization for Standardization (ISO) Standard No. 10993 and / or the United States Pharmacopeia (USP) 23 and / or the United States Food and Drug Administration (FDA) Blue Book Memorandum No. G95-1 entitled "Use of International Standard ISO-10993, Biological Evaluation of Medical Devices Part 1: Evaluation and Testing". Typically, these tests measure the toxicity, infectivity, pyrogenicity, irritancy, reactivity, hemolytic activity, carcinogenicity, and / or immunogenicity of the material. A biocompatible structure or material, when introduced into the majority of patients, does not provoke any serious adverse, prolonged, or increased biological reaction or response, as distinguished from the mild, temporary inflammation typically associated with surgery or implantation of a foreign body into a living organism.

[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 matricellular proteins. Examples of structural proteins include collagens (the most abundant proteins in the ECM), such as fibrillar collagens (types I, II, III, V, and XI), phasic collagens (types IX, XII, and XIV), short-chain collagens (types VIII and X), basement membrane collagens (type IV), and other collagens (types VI, VII, and XIII), elastin, and laminin. Examples of adhesive glycoproteins include fibronectin, tenascin, and thrombospondin. Examples of proteoglycans include heparin sulfate, chondroitin sulfate, and keratan sulfate. Examples of non-proteoglycan polysaccharides include hyaluronic acid. Matricellular 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 ECM, including thrombospondin (TSP) 1 and 2, tenascin, and SPARC (secreted protein acidic and rich in cysteine).

[0083] In certain embodiments, during decellularization (and other optional processing steps), not all of the naturally occurring lipids are removed from the lipid layer of the fish skin. Thus, the scaffold material can include one or more lipids from the fish skin, particularly the lipid layer of the fish skin. For example, the scaffold material can include up to about 25% w / w lipid (of the total dry weight of the scaffold material after lyophilization), 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 lipid. 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] The lipids in the scaffolding material can include, for example, fatty acyls (i.e., fatty acids, their complexes, and derivatives), glycerolipids, glycerophospholipids (i.e., phospholipids), sphingolipids, glycolipids, polyketides, sterol lipids (i.e., sterols), certain fat-soluble vitamins, prenol lipids, and / or polyketides. Examples of fatty acyls include saturated fatty acids, such as polyunsaturated fatty acids, fatty acid esters, fatty amides, and eicosanoids. In certain embodiments, the fatty acids include omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (found in high concentrations in fish oils). Other fatty acids found in fish oils 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, α-linolenic acid, γ-linolenic acid, behenic acid, erucic acid, and lignoceric acid. Examples of glycerolipids include mono-, di-, and tri-substituted glycerols such as monoacylglycerol, diacylglycerol, and triacylglycerol (i.e., monoglycerides, diglycerides, and triglycerides). Examples of glycerophospholipids include phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Examples of sphingolipids include phosphosphingolipids and glycosphingolipids. Examples of sterol lipids include cholesterol, steroids, and secosteroids (various forms of vitamin D). Examples of prenol lipids include isoprenoids, carotenoids, and quinones such as vitamins E and K, and hydroquinones.

[0085] The scaffolding material may contain one or more additional active agents (i.e., agents added during or after processing of the scaffolding material), such as antibiotics, antiseptics, 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 substances. The antibiotics, antiseptics, and antibacterial agents may be added in an amount sufficient to provide effective antibacterial properties to the scaffolding material. In certain embodiments, the antibacterial agent is one or more antibacterial metals, such as silver, gold, platinum, copper, zinc, or combinations 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. The anti-inflammatory agent may be added in an amount sufficient to reduce and / or suppress inflammation in the area of ​​the wound or tissue to which the scaffolding material is applied.

[0086] The scaffolding material can be used in a dry form. Alternatively, the scaffolding material can be rehydrated prior to use. In certain embodiments, one or more scaffolding materials are layered together to form a thicker scaffolding material.

[0087] Generally, the scaffold material is about 0.1-4.0 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 thick. The thickness may depend on a number of factors, such as the type of fish used as the starting material, processing, freeze-drying, and / or rehydration. Of course, if the product includes more than one layer of scaffold material, the thickness will be proportionately greater.

[0088] The shredded decellularized fish skin particles of the wound treatment and method embodiments advantageously provide a sterile, collagen-based matrix with properties that may promote regeneration, repair, and / or growth of tissue, such as endogenous tissue, while being configured to be formed or added to the wound to better conform 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 tunneled wounds, in a manner that is not available when using sheet-based materials. That is, the shredded decellularized fish skin particles may be configured to promote incorporation, i.e., cells and tissues of a patient being treated with the scaffold material may grow into the scaffold material, and the scaffold material may actually be incorporated / absorbed into the patient's body.

[0089] The shredded decellularized fish skin particles according to embodiments may be configured in embodiments to actively promote wound healing, such as by serving as a physical scaffold for infiltrating cells involved in wound healing / repair, such as cellular ingrowth and angiogenesis. The shredded decellularized fish skin particles of wound treatment embodiments are advantageously configured to retain the three-dimensional ("3D") structure of the decellularized fish skin, with an extracellular matrix ("ECM") recognizable, for example, for histological analysis. 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 an embodiment, the shredded decellularized fish skin particles have a maximum dimension within a range of a predetermined size threshold maximum and a size threshold minimum that is effective for preserving the matrix structure of the decellularized fish skin and promoting regenerative ingrowth 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, can be less than a maximum size, such as 1 mm, and greater than a minimum size, such as the size at which the ECM is destroyed. In an 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 the predetermined minimum and maximum size thresholds.

[0091] The chopped decellularized fish skin particles can be further configured to resist shear forces given their dimensions, thus allowing the chopped decellularized fish skin particles to provide improved wound care for patients who move or are moved between locations or environments, or during normal patient activities such as walking during recovery.

[0092] The shredded decellularized fish skin particles of the embodiments can advantageously be applied topically and / or implanted into a wound to provide a scaffold for cellular ingrowth and vascularization, and can further provide additional benefits including tissue scaffolding benefits such as adhesion barrier, soft tissue repair, and dehiscence prevention.

[0093] Colorant(s) Various examples of coloring agents are contemplated. In its broadest sense, a coloring agent, or a combination of coloring agents, that provides a color to the skin substitute that changes or disappears based on changes in conditions within the wound or changes to the skin substitute during the healing process. In a preferred embodiment, the coloring agent degrades upon attack by one or more proteases within the wound. With such a coloring agent, the coloring agent loses its color upon degradation by one or more proteases. For example, the coloring agent may provide a blue or purple color to the skin substitute. However, upon attack by one or more proteases within the wound after application of a wound treatment that includes a skin substitute and a coloring agent, the color of the skin substitute of the wound treatment is also degraded or disappeared, thereby changing the color of the applied wound treatment to the original color of the skin substitute or another color. However, the color change of the coloring agent is not limited thereto and may also include a color shift associated with changes in conditions within the wound. For example, the color provided by the coloring agent may be induced such that the original color of the skin substitute is not changed by application or addition of the coloring agent. However, a change in color of the colorant may be induced or caused by a change in conditions within the wound, thereby causing the skin substitute of the wound treatment to change to a new color, or a color different from the original color of the skin substitute.

[0094] Dyes may be used as colorants. A preferred example of a colorant is a thiazine dye, 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 textile dyeing, medicine, and research. It is used to treat methemoglobinemia at doses of up to 2 mg / kg over several hours.

[0096] Another embodiment of the colorant is a triarylmethane dye. An example of a preferred 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 hexamethylpararosaniline chloride, is a triarylmethane dye commonly used for histological staining in the Gram stain procedure. Topical gentian violet (V) is used to treat certain types of fungal infections in the mouth (thrush) and on the skin.

[0098] Another embodiment of the colorant is Brilliant Blue FCF (BB-FCF), which has the following structure: [ka]

[0099] Brilliant Blue FCF (BB-FCF), also known as Blue No. 1, is a triarylmethane dye used primarily as a blue colorant for processed foods, drugs, dietary supplements, and cosmetics. It is one of the oldest FDA-approved color additives and is generally considered to be non-toxic and safe.

[0100] Another embodiment of the colorant is indigo carmine (IC), which has 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 levels below 11.4 and yellow at pH levels above 13.0, and can further be used as a redox indicator, turning yellow when reduced.

[0102] Other dye chemistries or dye mixtures may be used and are contemplated by the inventors, including:

[0103] Woad Powder (HUE-3023), a wool dye proposed by The Woolery, has an INCI (International Nomenclature of Cosmetic Raw Materials) of Daiso extract. It is a dye chemical commonly used for yarn and garments, usually used for dyeing in alkaline environment. Woad Powder can be considered as a useful colorant due to the nature of the powder with the ability to bind to keratin proteins.

[0104] The color additive, D&C Green #5 Powder AN0725, is made from natural sources and is commonly used in cosmetics. The INCI name for this color additive is Green No. 5. The powder is a water-based dye in dry powder form. It can be selected as a colorant due to its typical water-based cosmetic color in powder form.

[0105] Color additive, Ultra Marine Blue H9-03R1, is used in cosmetics including eye makeup (not for lip products), soaps, and lotions. This color additive is based on natural resources and has the INCI name of Ultramarine Na6Al6Si6O24S4. This color additive can be an oil dispersible pigment, it is not soluble in water or oil, and has the CAS number: 57455-37-5. It is rated as being highly effective in cosmetics with strong dyeing power, and it is not soluble in water or oil when placed in cosmetics, so it can be selected as a coloring agent. However, this color additive can contain residues of undesirable substances, which must be taken into consideration.

[0106] Color additive liquid, FD&C Blue #1, is used in cosmetics, soaps, bath salts, and bath bombs. It is made from natural sources and has the INCI name of Blue No. 1. This color additive can be provided as a premixed water-based dye. It is a typical water-soluble liquid dye for cosmetics.

[0107] A color additive liquid, D&C Green #5, is also used in cosmetics, soaps, bath salts, and bath bombs. It is made from natural sources and has the INCI name of Green No. 5. This color additive can be provided as a premixed water-based dye. It is a typical water-soluble liquid dye for cosmetics.

[0108] A color 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 name of Green No.6 and Caprylic / Capric Triglyceride. This color additive is provided in premixed oil-based liquid dyes, blended for example in fractionated coconut oil, to extend shelf life. This dye can be considered as a product that reacts better with oil-based dyes, later withstands the washing process well, and does not dissolve on hydration. However, it should be noted that when using this dye, measures must be considered to reduce or address the permanent coloring of the wound, which may result in a tattoo effect for the patient.

[0109] Green Concentrated Food Coloring is a food coloring manufactured by Rayner. It has the INCI name Water, Tartrazine (E102) (1.87%), Brilliant blue FCF (E133) (0.13%), Acetic Acid. It is provided as a premixed water-based liquid food color mixture and therefore can be considered a colorant. The dye mixture is considered to be a non-toxic dye mixture.

[0110] Garnie natural Color, Mahogany Brown contains: Aqua, Deceth-3, Areth-12, Cocamide Mipa, Oleth-30, Ammonium Hydroxide, Deceth-5, Glycerin, Oleic Acid, Oleyl Alcohol, Hexadimethrine Chloride-2, 4-Diaminophenoxyethanol HCl, p-Aminophenol, m-Aminophenol, Ascorbic Acid, Hydroxyethylcellulose, Sodium Metabisulfite, Ethanolamine, Triticum Vulgare Oil, Thioglycerin, Polyquaternium-6, Toluene-2,5-Diamine, Polyquaternium-67, 2-Methyl-5-Hydroxyethylaminophenol, Ammonium Thiolactate, Simmondsia Chinensis Oil A hair colorant manufactured by Garnier having ingredients of jojoba seed oil, isopropanolamine, resorcinol, EDTA, and parfum. The dye is commercially available as a premixed hair color kit. This dye is another embodiment of a colorant since it is formulated to bind proteins and react with collagen in scaffolds or skin substitutes.

[0111] ELEA, Color & Care, Black, a hair colorant manufactured by ELEA. It has the ingredients Aqua, Cetelyl Alcohol, Ammonia Ceteleth-20, Cetrimonium Chloride, Cocamdopropyl Betatan, Oleic Acid, Propylene Glycol, PEG-40, Hydrogenated Histrole Oil, p-Phenylenediamine, 2,4-Diaminophenoxyethanol, HCl, Vitis Vinifera Seed Oil, Sodium Metabisulfite, Erythorbic Acid, Parfum, Coumarin, Limonene, Linalool, Resorcinol, 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] Although various examples and embodiments of colorants are provided herein, this description of possible colorants, either as dyes or color additives, is not intended to be exhaustive of all possible colorants and should not be considered as such.

[0113] Adding colorants to skin substitutes In a preferred embodiment, methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV) are used as the coloring agent added to the skin substitute.

[0114] In embodiments using a combination of methylene blue (MB) and gentian violet (GV), the methylene blue (MB) and gentian violet (GV) are used together in equal weight ratios. However, in other embodiments, the methylene blue (MB) and gentian violet (GV) are used together in different weight ratios. Other embodiments include any combination of two or more of these dyes, methylene blue (MB) and gentian violet (GV), and other dyes. Exemplary methods and embodiments are described below.

[0115] In a first embodiment, a decellularized fish skin scaffold 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 scaffold material made from minimally processed skin of wild-caught Atlantic cod native to Iceland is referred to as the "scaffold" or "scaffold material" provided as an embodiment of the skin substitute.

[0116] Below is a description of methods for adding one or more colorants to the scaffold to enhance the fastness of the colorant(s).

[0117] In a preferred embodiment, a common process for adding methylene blue (MB) and / or gentian violet (GV) staining agents to the scaffold is used.

[0118] An exemplary procedure used 100 mL of dye solution based on either deionized water or phosphate buffered saline (hereafter 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). Freeze-dried scaffold pieces with dimensions of approximately 4 x 4 cm and weighing 0.25-0.30 g were added to the solution and left for 3 h. The Kroma scaffolds were then removed from the solution and washed with tap water, followed by rinsing with deionized water and freezing. The resulting dyed scaffold 300 is shown in Figure 3.

[0119] When MB and GV are used as described above, the total amount of both dyes in the scaffold is about 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 at the same total concentration (0.002 wt%) or per dye concentration (0.001 wt%) in water or PBS solution. The same total concentration is obtained in the scaffold for any single dye or other combination of dyes listed above even if the total concentration or volume of the dye solution is changed slightly. A UV-VIS spectrophotometer can (and was used by the inventors) measure the absorbance of the colored solution before and after the dyeing process with any single dye or any combination of dyes, and to some extent the concentration, to determine the affinity for adsorption to the scaffold.

[0120] 4A, 4B, and 4C show the resulting scaffolds containing 0.001 wt% MB in scaffold material 410 of FIG. 4A left for 24 hours, 0.001 wt% GV in scaffold material 420 of FIG. 4B left for 24 hours, and 0.001 wt% total MB / GV in a 25 / 75 blend ratio in scaffold material 430 of FIG. 4C left for 24 hours, stained with 100 mL of dye solution based on either deionized water or phosphate buffered saline (hereinafter abbreviated as "PBS").

[0121] In other embodiments, other additional combinations of dyes were used, including: (1) BB-FCF and IC applied together (when MB and GV are applied) using the same experimental setup as above, (2) BB-FCF and / or IC applied before and after MB and / or GV to extend the life span in 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 discussed in later sections herein. Figure 5 shows the resulting scaffold 510 stained with BB-FCF and IC applied by weight similar to the above examples with stains MB and GV as above, with 0.001% by weight of each stain BB-FCF and IC, with a total concentration of 0.002% by weight, left for 3 hours.

[0122] Alternative method using a mordant In other embodiments, various methods or color fasteners are used to enhance the fastness of the colorant or combination of colorants added to the scaffolding material.

[0123] Mordants, or dye fasteners or fixatives, are a group of compounds used in the biological dyeing and textile industries, consisting mainly of divalent metal salts. Compounds such as tannic acid and cream of tartar (the potassium salt of tartaric acid) are also commonly used, often for the same purpose, but they are generally not considered true mordants.

[0124] The choice of mordant often depends on the dye being used, for example some zinc salts may be used with MB, iodine (KI+12) may be used with GV. Iodine is also used as a mordant in Gram staining, but is considered to be a scavenger rather than an actual mordant.

[0125] One definition of a mordant is a polyvalent metal ion that forms a coordination complex with certain dyes, although this definition is not necessarily limited to the term "mordant" in this disclosure, and as reflected above, some compositions (e.g., tannic acid, cream of tartar, iodine) are generally considered by those skilled in the art to be mordants even though they do not fit this definition.

[0126] Mordants can be applied to the coloring process in three general ways: (1) the substrate is treated with the mordant and then with the dye, pre-mordant (on-chrome); (2) the mordant is present in the coloring solution from the beginning, meta-mordant (metachrome) (this process is simpler than pre-mordant / post-mordant but is only applicable to a few dyes); and (3) the substrate is first treated with the dye and then with the mordant, after-mordant (afterchrome).

[0127] 6A and 6B show mordanted scaffold materials, where FIG. 6A shows a post-mordanting sample of Kroma scaffold material 610 dyed with alum at a concentration of 0.002% by weight with a combination of MB / GV, and FIG. 6B shows a pre-mordanting sample of scaffold material 620 dyed with alum at a concentration of 0.002% by weight with a combination of MB / GV. Alum, also known as aluminum sulfate, is one of the most used mordants in textiles, as it enhances the brightness and saturation of colors, besides providing good fastness to various dyes. However, it is by no means the only mordant that can be used with the scaffold. In other embodiments, possible mordants / salts include, but are not limited to, NaCl, MgCl2, MgSO4, CaCO3, CaCl2, KCl, ZnCl2, some other Zn salts, or KI / I2 are also used.

[0128] In another embodiment, a meta-mordant variation is shown in Figures 6C and 6D, where scaffold material 630 is dyed with a combination of MB / GV at a total concentration of 0.002 wt% in Figure 6C, where 0.5 grams of CaCl2 was added, and in Figure 6D, scaffold material 640 is dyed with a combination of MB / GV at a total concentration of 0.002 wt% in which 0.5 grams of NaCl was added.

[0129] In embodiments, any of these metal salts / mordants, or a combination of two or more, are used with any dye or any combination of dyes listed above, or any other dye combination and coloring technique.

[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 for more than 48 hours at room temperature. In our case, our experimental results show that heating the scaffold in sodium chloride solution at approximately 80°C for 2 hours can substantially degrade collagen into a more gel-like form, possibly due to partial collagen degradation into gelatin. Therefore, the "cooling method" or the hybrid method at approximately 37°C for 12 hours is preferred.

[0131] Meta-mordants are generally considered to be the most limiting method. This is due to a variety of factors, including the solubility of the dye-mordant complex formed during the process (known as a dye lake). The solubility of the complex is often lower than that of the mordant and dye, causing them to precipitate separately, limiting the mordant-dye combinations that can be applied. Furthermore, when using meta-mordants, the time in solution depends on the mordant time, which is approximately two days at room temperature. Thus, the dyeing process may need to be longer or at a higher temperature. The amount of color adsorbed has been shown to be directly related to the time in solution, and possibly also correlated to temperature.

[0132] Preferred embodiments include pre-mordanting or post-mordanting, where post-mordanting is potentially more suitable for our application, considering that the scaffold does not lose much color during the mordanting process, since post-mordanting can be used without modifying the current coloring and quantification methods. The pre-mordanting process may change the adsorption rate of the dye, since the dye lake is formed directly on the surface of the scaffold during adsorption. Furthermore, the mordant may "leak" into the dye solution, which may cause precipitation of the dye molecules or a change in the absorbance intensity. In either case, this may cause problems when measuring the amount of dye adsorbed on the scaffold.

[0133] As previously mentioned, alum is the preferred mordant, however, in other embodiments, other mordants may be used, including but not limited to metal salts such as sodium, magnesium, potassium, and iron salts.

[0134] pH Gradient Staining or "Through Staining" In another embodiment, a process of "through-dyeing" was used for the staining treatment of the scaffolds. This process involves a stepwise change in pH of the dye solution during the staining process from a slightly basic pH of 9-10 to a slightly acidic pH of 3-4. This can be achieved using various weak acids / bases (e.g., acidic acid / sodium bicarbonate), dilute solutions of strong acids / bases (e.g., HCl / NaOH), or very small amounts of concentrated strong acids / 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 effectiveness of through-dyeing is believed to be due to the limited stability of the tertiary structure of the protein (in this case, collagen) over the range of the pH scale. When exposed to a pH that the protein is not developed to handle, the protein structure deforms by opening up, exposing the binding sites for the dye.

[0135] In Fig. 7, the various resulting scaffolds with pH grading in the dyeing process are shown. First, a scaffold 710 is shown after dyeing with a combination of MB / GV at a total concentration of 0.002 wt% when a pH gradient is applied. A scaffold 720 is shown after dyeing with MB at a total concentration of 0.002 wt% when a pH gradient is applied. A scaffold 730 is shown after dyeing with a combination of MB / BB-FCF at a total concentration of 0.002 wt% when a pH gradient is applied. And a scaffold 740 is shown after dyeing with BB-FCF at a total concentration of 0.002 wt% when a pH gradient is applied.

[0136] Alternative staining or fixation methods In other embodiments, non-traditional staining or color fixing methods are used. Below is a description of some non-traditional staining 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 described above (MB, GV, BB-FCF, IC) are not only soluble in water, but also soluble in various solvents such as ethanol, and are slightly lipophilic.

[0138] In some embodiments, MB and / or GV are dissolved in ethanol and freeze-dried scaffolds are dyed in the ethanol solution. This results in much lighter coloring when compared to similar aqueous methods, even when more concentrated dye solutions and longer dyeing times are applied. Although these embodiments visually show brighter colors overall, these embodiments may still be considered effective and preferred, as they may tend to improve color fastness and reduce the possibility of permanent wound tattooing, while still providing an effective colored skin substitute.

[0139] In other embodiments, the dyes are dissolved in oleic acid, but in these embodiments, their solubility is lower. However, a 70 / 30 oleic acid / ethanol mixture was used, resulting in a higher solubility. It was found that the resulting scaffolds were generally darker in color than ethanol and oleic acid at the same time and dye concentration.

[0140] In other embodiments, vegetable oils have also been used. Also, fish oil / cod liver oil can be used. The use of oil / organic solvent based dye solutions can be done with any combination of dyes and can also be done on scaffolds pre-mordanted with various oils, fatty acids, their salts, and solvents.

[0141] In other embodiments, a coating treatment was performed after dyeing. Of these embodiments, those of most interest are oil and sugar based coats.

[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 the scaffold after dyeing. The samples acquired a certain degree of resistance to in vitro decolorization and degradation experiments compared to similar samples without coating. Furthermore, this could also be done using appropriate fatty acid alkyl esters.

[0143] In other embodiments, sugar-based coats are made with a variety of sugars, either monosaccharides such as ribose, fructose, or dextrose, or disaccharides such as sucrose or maltose. The sugar of choice is dissolved in an aqueous solution and freeze-dried again after soaking the scaffold. Non-reducing sugars may be dissolved in the coloring solution. Sugars may increase the stability of the collagen itself by introducing additional crosslinks. In addition, sugars contain a large number of -OH groups, which may facilitate further binding to dye molecules through hydrogen bonds or dipole forces. Furthermore, nitrogen-containing sugars such as N-acetylglucosamine may form covalent bonds with the free amino / acid termini of collagen and certain dyes.

[0144] Nearly all possible methods and embodiments described herein above may be used together, however, the use of multiple components of each category, e.g., two or more mordants, to increase color vibrancy, fastness, etc., may also increase the complexity, possible side effects, and overall cost of producing the dyed scaffold.

[0145] Thus, the preferred method and embodiment to generate a suitable prototype is similar to the "basic" dyeing process. The most notable problem identified is the dye's longevity in in vivo conditions (e.g., in mice). Possible improvements could be to increase the binding of the dye molecules in the collagen matrix of the scaffold using a mordanting step, a pH gradient, or a combination of the two. As shown in Figure 8, two preferred embodiments of dyed scaffolds are shown for comparison. Scaffold 810 is dyed using a combination of MB / GV at a total concentration of 0.002 wt%, and scaffold 820 is dyed using a combination of MB / GV and a pre-mordanting dye at a total concentration of 0.002 wt%.

[0146] Collagenase-catalyzed degradation of scaffolds When the scaffold is used as a biological bandage, the body breaks down the large scaffold into a "pool" of microscopic fragments, which aids in the reconstruction and growth of the affected area. In validation experiments, a PBS solution of collagenase was chosen to mimic this scaffold degradation process and its effect on the pigment(s) in the scaffold sample. In nature and in humans, the main function of collagenase is to break down collagen to the peptide level, which occurs in damaged tissues, for example in the skin, helping the body generate new healthy tissue.

[0147] For this, a stock solution of collagenase at 0.50 mg / mL in PBS was prepared. In initial experiments, the stock solution was diluted to 10 μg / mL or 100 μg / mL. A total of nine solutions were made (10 ml) using either PBS or "human plasma-like solution" as the bulk of the solution, after which scaffold pieces were placed in the solutions and kept at room temperature for extended periods.

[0148] We found that unstained scaffolds began to degrade in collagenase PBS solution. In the case of plasma-like medium, the solution appeared to inhibit collagenase, as seen by the comparative levels of scaffold degradation after approximately 3 days, even though the concentration of collagenase was 10 times higher in the plasma solution. During this time, no significant changes were observed in scaffolds stained with a combination of MB and GV at a concentration of 0.002%.

[0149] To degrade the MB / GV staining, a fairly high concentration of collagenase was applied. For this, the original 0.5 mg / mL PBS solution was used. After testing unstained scaffold pieces for comparison of time and degradation level, a series of variations were tested. It was found that the complete degradation to fine particles only took approximately 24 hours. Turning to the stained samples, as shown in Figure 9A, a first scaffold sample 910 stained in a 0.001 wt% solution of MB / GV was tested. With a 0.5 mg / mL collagenase solution, the complete degradation of that sample took approximately 2 days. As can be seen in Figure 9B, some of the color has bled into the solution, but most of the dye is still bound to the small collagen particles 920.

[0150] The next five "prototypes" described in the previous section were tested in the same manner. The prototypes tested were: 1) 0.002 wt% MB / GV in water, 2) 0.002 wt% MB / GV in PBS, 3) 0.002 wt% MB / BB FCF in PBS, 4) 0.001 wt% IC in water, and 5) stained scaffolds with 0.002 wt% MB / GV in water coated with a mixture of triglycerides, monoglycerides, and free fatty acids derived from fish oil. The samples were checked over the next four days, and in all cases except one (sample 4: 0.001 wt% IC in water), the total degradation took four days.

[0151] These experiments showed that the stained scaffolds (1-5 above) degraded into fine fragments and in all cases, a significant portion of the dye remained on those fragments. This suggests that the dye is tightly bound to the collagen / peptides of the scaffold and not just to the surface. As mentioned above, in all but one case (i.e., sample 4: 0.001 wt% IC in water), degradation took approximately 4 days and no significant differences were observed between the samples stained with MB / GV. Samples 3 and 4 were slightly different, with sample 4 stained with IC being almost completely degraded in just under 24 hours, leaving only a few fragments. Sample 3 was more stable compared to sample 4, but degraded faster and into smaller fragments than the other three samples.

[0152] In the above embodiments, the coloring of the scaffold generally involved combining methylene blue (MB) and gentian violet (GV) in equal weight ratios. However, this is not necessarily the case. The amount of dye can be adjusted to produce a scaffold with a specific amount of colorant, with the goal of the amount of colorant being below, and in some embodiments well below, the maximum allowable 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 allowable amount can 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 colorant uses a 100 mL dye solution (based on deionized water or PBS) containing 0.001% by weight of each colorant (totaling 0.002% by weight or 20 mg / L). However, this amount of colorant in the dye solution can be increased or decreased to suit the needs of the skin substitute to which the colorant is applied. For example, the dye solution can be 1.0-10.0% by weight of colorant or colorant (based on deionized water, PBS, or some other dye solvent), 1.0-20.0% by weight of colorant or colorant (based on deionized water, PBS, or some other dye solvent), 1.0-0.01% by weight of colorant or colorant (based on deionized water, PBS, or some other dye solvent), depending on the colorant, skin substitute to which the colorant or colorant is being added. The dye or colorant may have an amount of 0.01 to 0.001 weight percent colorant or colorant (based on deionized water, PBS, or some other dye solvent), 0.05 to 0.002 weight percent colorant (based on deionized water, PBS, or some other dye solvent), 0.01 to 0.0002 weight percent colorant (based on deionized water, PBS, or some other dye solvent), or 0.01 to 0.0002 weight percent colorant or colorant (based on deionized water, PBS, or some other dye solvent).

[0154] A piece of scaffold with dimensions of 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 noted above, the size of the scaffold material and the time the scaffold material is left in the staining solution may be altered. Additionally, the size of the scaffold material may of course be altered and necessary adjustments may be made to the volume and concentration of the dye solution. The scaffold is then removed from the solution and washed with tap water, followed by rinsing with deionized water, freezing, and freeze-drying or lyophilization.

[0155] In some embodiments, it has been found that the relative amounts of MB and GV adsorbed by the scaffolds change when PBS is used instead of deionized water as the base of the solution, from approximately 60% GV and 40% MB by weight for the aqueous solution to approximately 40% GV and 60% MB by weight for PBS. However, the total amount of colorant adsorbed remains approximately 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 may range from 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 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, in other embodiments, food dyes, or more precisely the active compounds (dyes / pigments) in food dyes, are used in combination with or in place of MB and GV.

[0157] In one embodiment, one fat-soluble and one water-soluble food coloring are used. The dye in the fat-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 was approximately 40% by weight of the food coloring, while the rest of the additives were for "fat-soluble". The dye in the water-soluble coloring was E132, i.e., indigo carmine (IC), which was approximately 85% by weight of the food coloring.

[0158] It was discovered by the inventors that only the food dyes could be removed from the scaffold material by both enzymatic degradation using collagenase and by placing it in approximately 1 M sodium bicarbonate solution.

[0159] The binding mechanism of the above-mentioned colorants found to be bound to the collagen / peptide of the scaffold material allows binding to be effected to other collagen or peptide based skin substitutes as well as surfaces via similar binding mechanisms using similar or suitable colorants.

[0160] The scaffold 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 can be round fish such as cod, haddock, and catfish, flatfish such as halibut, plaice, and flounder, salmonids such as salmon and trout, mackerel such as tuna, or small fish such as herring, anchovy, mackerel, and sardine. Furthermore, other collagen, peptide, 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] Testing in mice and patients and results mouse An embodiment of a decellularized fish skin scaffold material made from minimally processed skin of wild-caught Atlantic cod native to Iceland was provided as a skin substitute. Again, in the following subsections, unless otherwise specified, the "fish skin" used as a scaffold material made from minimally processed skin of wild-caught Atlantic cod native to Iceland will be referred to as "fish skin" as a "scaffold" or "scaffold material" provided as an embodiment of a skin substitute.

[0162] A total of 52 mice were tested using embodiments of the pigmented scaffold material as a skin substitute.

[0163] In the first pilot, pilot 1, four mice were treated.

[0164] Pilot 1 included: 1) Fresh decellularized fish skin stained with a colorant of 0.005 wt% MB + 0.005 wt% GV in water for 3 h before freeze-drying; and 2) Fresh decellularized fish skin stained with a colorant of 0.010 wt% MB + 0.010 wt% GVs in water for 3 h before lyophilization.

[0165] In the second pilot, pilot 2, 16 mice were treated.

[0166] Pilot 2 included: 1) Fresh decellularized fish skin stained with 0.001 wt% MB + 0.001 wt% GV colorant, immersed in cold sugar solution and dyed in water for 24 h before freeze-drying; 2) 0.001 wt% MB + 0.001 wt% GV colorant, freeze-dried fish skin dyed in water for 3 h before immersing in cold sugar solution and freeze-drying; 3) 0.001 wt% MB + 0.001 wt% GV colorant, freeze-dried fish skin dyed in water for 3 hours before being immersed in mineral oil and freeze-dried; and 4) Freeze-dried fish skin dyed with 0.001 wt% MB + 0.001 wt% GV colorant in water for 3 hours before freeze-drying.

[0167] In the second pilot, pilot 3, 32 mice were treated.

[0168] Pilot 3 included: Colorant: 0.001 wt% MB + 0.001 wt% GV, freeze-dried fish skin stained in PBS for 3 h before freeze-drying.

[0169] The results of each of the Pilot 1, Pilot 2, and Pilot 3 mouse studies yielded the following results: No unexpected inflammation or other adverse events were detected after using the pigmented fish skin as a scaffolding material. The treatment product (scaffolding material) degraded in the normal time and the wound healed normally. Importantly, no permanent or semi-permanent marking of the wound bed was detected.

[0170] Human patients Three patients (Patient 1, Patient 2, and Patient 3) were treated with minimally processed skin of wild-caught Atlantic cod native to Iceland, referred to in this subsection as “fish skin,” “scaffold,” or “scaffold material.”

[0171] In each of the three patients (Patient 1, Patient 2, and Patient 3), scaffold materials were generated similarly to Pilot 3 above, using a staining agent of 0.001 wt% MB + 0.001 wt% GV, freeze-dried fish skin stained in PBS solution for 3 h 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 7 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 of patient 3 were treated with pigmented fish skin from January 20, 2022 to February 10, 2022, and Figures 12A-12N show the treated wounds each time the wound dressing was changed. Figure 12A shows the pigmented fish skin applied on day 0, and Figure 12B shows the same wound on day 4. Freshly dyed fish skin was 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, freshly dyed fish skin was applied to a different wound in Figure 12E, Figure 12F shows the same wound after two days, and Figure 12G shows the same wound after five days. Figure 12H shows the application of freshly pigmented fish skin, Figure 12I shows the results after two days, and Figure 12J shows the results after four days. Finally, FIG. 12K shows the application of freshly pigmented fish skin, FIG. 12L shows healing after 2 days, and FIG. 12M shows the healing results after 4 days.

[0175] In each of the above patients 1 to 3, no device-related inflammation or other adverse events were reported after the use of the pigmented fish skin. The applied treatment appears to promote healing of these chronic wounds. Furthermore, the applied pigmented fish skin degraded normally in the wound. Also, no permanent or semi-permanent marking of the wound bed was detected after the 5th day.

[0176] Further examples As discussed above (e.g., when using Kerecis™ fish skin derived cell scaffold products to heal wounds as disclosed in U.S. Pat. No. 8,613,957), the inventors have discovered significant challenges where clinicians unwittingly err or otherwise struggle to distinguish between a wound healing scaffold and an infection. This may be due, at least in part, to the color and / or odor associated with the wound healing scaffold as it degrades and begins to integrate into the surrounding tissue, which may sometimes have a color similar to infected tissue (e.g., purulent infection) and may be mildly aromatic, thus interpreting by some as an odor similar to infected tissue. Thus, the inventors have discovered that challenges exist in the art that could greatly benefit from improved products or improvements to known products.

[0177] One solution is to pseudocolor the fish skin derived cell scaffold so that it can be more easily identified in the clinic and / or differentiated from the surrounding tissue when placed in a wound bed. To that end, the following disclosure provides exemplary data from a series of studies focused on identifying colorants that can remain stable over time and that can be incorporated into the fish skin scaffold during the processing / manufacturing process.

[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 cellular scaffold products, which are made from minimally processed wild-caught Atlantic cod skin 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 (e.g., as listed in Table 1). 50 mL of Decell solution was aliquoted into each of seven separate plastic tubes that could be secured with their respective lids. The first tube contained only Decell solution and served as a control. A 0.5 mL aliquot of each of the six prepared color solutions was added separately to the corresponding tube containing 50 mL of Decell solution. The mixtures were monitored over time for any reaction or visible changes.

[0181] After 30 minutes and 24 hours of incubation, the solutions in each tube containing each stain were monitored and photographically documented at the start of the experiment.

[0182] Many of the colorants were found to be quite bright in the Decell solution at the beginning. In the first 20 minutes, most of the colorants began to fade, with the notable exception of methylene blue. This trend continued, and after 24 hours, all of the colored Decell solutions, except for the Decell solution with added methylene blue, turned white or nearly white. Therefore, methylene blue color is believed to be the preferred embodiment of the colorant added during the decellularization stage of the manufacture of Kerecis™ fish skin-derived cellular scaffold products, which are made from minimally processed skin of wild-caught Atlantic cod, as described in U.S. Pat. No. 8,613,957.

[0183] In another embodiment, a method 1300 of treating a wound using a tissue regeneration wound treatment is provided, as shown in Figure 13. At step 1310, a tissue regeneration wound treatment is provided that includes a skin substitute and a colorant, the colorant being a biocompatible colorant that degrades upon protease attack within the treated wound. At step 1320, the tissue regeneration wound treatment is applied to the wound bed. And at step 1330, it is determined whether the skin substitute has been degraded within the wound by protease attack by determining a color change in the colorant.

[0184] In a further exemplary method, a tissue regeneration wound treatment including a skin substitute in the form of a blue-colored (e.g., MG / GV) extracellular matrix 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 upon wound inspection. If the color is blue, the tissue regeneration wound treatment is considered (correctly) intact and it is concluded that cellular ingrowth has (correctly or probably) occurred. If the wound treatment is no longer blue, it has sloughed off and needs to be washed off and new material applied to the wound bed.

[0185] The coloring material used must be biocompatible and degrade as proteases attack the matrix itself. It does not have to be permanent, and may leave a permanent color or "tattoo effect" on the wound after healing has taken place.

[0186] Additional Testing The first color test was performed on de-hulled fish skin. Tests were performed on a fish skin-based wound product to see how the material would respond to various dye chemistries. The purpose was to see how the fibrous collagen material would react to various stains and if it would react differently under wet and dry conditions.

[0187] Test Scheme Glass bowls, tweezers, and airtight plastic containers were used for the experiments. These tests were to answer the questions of how the collagen material reacts with different types of dyes, whether oil-based or water-based reacts better with the protein, whether it is retained through washing, and at what point in production the dyeing of the molted fish skin scaffolds is optimal. The various dyes / colorants / pigments / color additives tested included Woad 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 concentrated food coloring; and Garnier Natural Color, Mahogany Brown.

[0188] Coloring before freeze-drying In the first step, the decellularized fish skin is colored before being freeze-dried. This is done to see how the material reacts with the color when wet, and how the colorant reacts upon washing and freeze-drying. Tests were performed after the decellularization step in the manufacture of decellularized fish skin wound products.

[0189] The decellularized fish skin scaffolds were kept in the dye chemicals for 60 min and then washed in continuous running water for 2 h.

[0190] Coloring after freeze-drying The second step of the test involved dyeing the material after it had been freeze-dried. This was to see if there was any difference in how the scaffold reacted with the color after freeze-drying, and if the structure was more open to dye chemicals. The sheets were then freeze-dried again.

[0191] Test procedure A piece of skin was taken from the decellularized fish skin and cut into small pieces. The pieces were placed in colorants, one part neat, one part a mixture of coloring powder and water / oil, or a mixture of colorant and hair color developer. The pieces were left for 2 hours, after which they were thoroughly washed, inspected, and photographed. The pieces that looked promising were immersed in water in a closed container and stirred until the next morning. This was to see if the color would eventually become insoluble in water. All pieces were inspected again and washed again. After immersion in pure water, all solutions were colored after 5 minutes. The promising pieces were sent for freeze drying (frozen at -80°C) and lyophilized in a freeze dryer.

[0192] A better understanding of various embodiments of the present disclosure may be had from the following description taken in conjunction with the accompanying drawings, in which like reference numerals refer to like elements and in which:

[0193] While the disclosure is susceptible to various modifications and alternative constructions, specific exemplary embodiments are shown in the drawings described below. It is to be understood, however, that there is no intention to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention of the present invention is to cover all modifications, alternative constructions, combinations, and equivalents falling within the spirit and scope of the present invention.

[0194] The references used are provided for convenience only and therefore do not define the scope of protection or embodiments.

[0195] Unless a term is expressly defined in this application to have a stated meaning, it is understood that there is no intention, either explicitly or implicitly, to limit the meaning of such term beyond its plain 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" broadly includes medical care and / or medicine 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 be medical care provided (i.e., in the form of a method or series of prescribed actions) or it can refer to medicine used to preserve or impart certain properties to something.

[0197] As a non-limiting example, the particulate form of the decellularized fish skin disclosed herein can be referred to as a "treatment," i.e., a medicine that can be used to preserve and / or stabilize a wound or provide any of the other disclosed beneficial effects to the wound site. Similarly, in some examples, the treatment includes the use of the disclosed decellularized fish skin in particulate form in a method for stabilizing and / or protecting a wound.

[0198] The terms "decellularization", "decellularized fish skin", "acellular fish skin", and the like, as used herein, refer to fish skin made by any method, including any of the embodiments disclosed in U.S. Patent No. 8,613,957, entitled "Scaffold Material for Wound Care and / or Other Tissue Healing Applications". Thus, the terms "decellularization", "decellularized fish skin", "acellular fish skin", and the like, as used herein, include fish skin from which a significant amount of cellular and nucleic acid content has been removed, leaving behind a complex three-dimensional stromal structure of extracellular matrix material (ECM). Generally, decellularization as described above is a gentler form of treatment than is otherwise required and / or routinely performed on mammalian tissues, which often utilizes 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 production of a scaffolding material that maintains the three-dimensional structure of the native extracellular matrix components, which in some instances provide a physical medium through which stem cells and other cells that contribute to the wound healing process can migrate across and / or be supported to promote wound healing. The native structure of extracellular components such as collagen is maintained within the decellularized fish skin scaffolding material, in addition to other native components such as omega-3 polyunsaturated fatty acids (PUFAs).

[0200] Other scaffold materials derived from mammalian skin / membrane, such as placenta-based wound treatments, may be used as skin substitutes.

[0201] Skin substitutes based on decellularized fish skin are preferred because the risk of disease transmission from Atlantic cod (Gadus morhua) and many other fish species to humans is nonexistent, or at least much less likely. Furthermore, decellularized fish skin tends to be free of allergy-causing components, greatly reducing the risk of allergic and other immune reactions. To reduce the 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, decellularized fish skin provides a natural scaffold to promote wound healing, since although skin cells are stripped during processing, it maintains the native three-dimensional structure of the extracellular components. In contrast, mammalian scaffold materials lack three-dimensional structure and are missing other native extracellular components, and are unable to promote wound healing in the same manner 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 reconstituted collagen materials are preferably not harvested through harsh physical and chemical treatments that fail to maintain their native three-dimensional structure, especially in the context of other natural extracellular components. As with the mammalian-derived scaffold materials discussed above, the lack of native structure and / or three-dimensional extracellular matrix environment provided by reconstituted collagen materials may reduce the effectiveness of the skin substitute in promoting wound healing. Of course, the selection of a skin substitute must take into account the cost and consistency of the skin substitute's production, as well as other factors, and in some cases or applications the use of such reconstituted collagen materials may actually be preferred.

[0203] Additional considerations regarding the onset of infection Wound treatments are often necessarily administered in austere environments, e.g., at or near the site of injury in combat situations, by non-medically trained personnel. The inventors have discovered a critical need for wound treatments, e.g., broad antimicrobial spectrum antimicrobial activity with the tissue regeneration capabilities, bacterial barrier, and analgesic properties of fish skin grafts. The inventors have discovered that wound treatment products that are easy to store and carry and can function as either definitive or temporary treatments would be particularly helpful, e.g., by reducing the need for evacuation of wounded individuals in combat or emergency situations.

[0204] As mentioned above, infection is a major challenge in emergency and combat wound management. It is responsible for morbidity and mortality of military personnel in the wounded or battlefield. For example, infection accounts for one-third of all casualties, prolonging treatment and increasing the risk of amputation. Due to the unique mechanism of injury and the harsh environment, combat wounds are prone to contamination and more difficult to treat. An early sign of infection is bacterial imbalance within the wound. Common pathogens found in wounds at early stages include both Gram-positive (G+) and Gram-negative (G-) strains. Once infection occurs, emergence of Gram-negative and multi-drug resistant (MDR) bacteria is observed. Thus, the inventors have identified a strong need for effective and immediate interventions to reduce the risk of infection to benefit soldiers and emergency personnel.

[0205] The tissue regeneration wound treatment of the present disclosure, which in some embodiments may be a blue antimicrobial fish skin graft, provides a novel visual cue 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, however, the wound treatment of the present disclosure is impregnated with antimicrobial agents in the form of antimicrobial colorants such as methylene blue (MB) and gentian violet (GV) or as an additional additional active agent. The wound treatment of the present disclosure integrates into the wound bed over time and releases 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 permanently discoloring the wound surrounding tissue.

[0206] Conventional field dressings available in combat or emergency environments provide fast-acting coverage, are field deployable in austere environments, can be used by the patient or a companion, and can often be used with saline to rinse and dehydrate. However, conventional field dressings do not provide broad antimicrobial coverage, must be changed daily, do not integrate into the wound bed, do not enhance wound healing, and do not provide a visual aid for integration of self-care with the care of others.

[0207] Antimicrobial silver dressings, which may also be used in combat or emergency settings, may provide fast-acting coverage, be field deployable in austere environments, may be usable by the patient or a companion, and may provide broad antimicrobial coverage. However, antimicrobial silver dressings cannot be used with saline for rinsing and rehydration, must 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 of integration for self-care or the care of others.

[0208] In comparison, the wound treatment of the present disclosure provides a fast-acting coverage, is field deployable in harsh environments, can be used by the patient or a companion, and provides broad antimicrobial coverage. Additionally, the wound treatment of the present disclosure can be used with saline for rinsing and rehydration, requires replacement (based on a color visual aid) after 5-10 days, and significantly, the wound treatment of the present disclosure integrates into the wound bed, enhances wound healing, and provides a suitable and effective visual aid for integration of self-care with the care of others.

[0209] The wound treatment of the present disclosure is well suited for a combat or emergency environment because it considers and addresses the needs of soldiers and medical personnel: Antibacterial activity: Methylene blue MB is a potent antibacterial dye against G- bacteria. It reduces the bacterial burden in wounds and reduces hypergranulation. GV is an antibacterial dye against G+ bacteria and can affect proinflammatory mediators; Shelf life: The wound treatment is stable for more than 3 years at room temperature and robust to shock. Stability at high temperature and humidity for long periods of time is being tested; Packaging: The wound treatment in the preferred embodiment is individually packaged in vacuum sealed military grade foil pouches containing sheets of dry, sterile fish skin. The pouches are small, lightweight and fit easily into a pocket or medical bag (100cm2 fish skin, 2g). 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 use. The colorant assists the user to distinguish between wound pus / shedding and fish skin integrated into the wound bed, allowing for straightforward follow-up care; Non-staining: Wound treatments use medical grade color compounds with known coloring and degradation profiles. No staining was observed with normal topical use. If any pigment is absorbed, the degradation profile was found by the inventors to be 6-12 days; Removable: There is no need to remove the wound treatment from the wound. The skin substitute, such as fish skin, recruits natural human cells into its structure, where the cells eventually transform the skin substitute, such as fish skin, into new tissue. However, if desired, once the fish skin begins to integrate, the product can be easily removed by lifting it with tweezers or wiping it off with damp gauze; Use in harsh environments: Wound treatments can be used at or near the injury site as definitive wound treatment or temporary antibacterial covering. Skin substitutes such as fishskin integrate slowly, resulting in less frequent dressing changes; Pain reduction: Wound treatments create their own internal environment by covering the wound with skin. In the case of fish skin, the graft is rich in fatty acids, including omega-3, which help shield exposed nerve endings, reduce inflammation, and positively impact pain via lipid mediators.

[0210] A significant objective of the present disclosure is to provide the Department of Defense and emergency personnel with an innovative solution for wound management at or near the injury site as an FDA approved antimicrobial skin substitute. The wound treatment of the present disclosure provides excellent healing properties along with strong antimicrobial activity. The wound treatment can be applied as definitive care for smaller, less severe wounds and as a temporary antimicrobial cover for severe injuries requiring transition to higher levels of care. Additionally, the coloring agent helps medical providers distinguish between the integrating skin substitute and pus or slough tissue of the wound.

[0211] The wound treatment of the present disclosure promotes wound healing through a combination of the following approaches: 1. Acts 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 lowering the risk of infection. 3. Fewer dressing changes result in less exposure of the wound to contamination and mechanical trauma from repeated dressing removal. 4. Color is a guide to non-medically trained users in optimal dressing and antimicrobial management. 5. Biomolecules naturally present in skin substitutes, e.g. fish skin (omega-3 and collagen), or additional active agents reduce pain, inflammation, and bleeding.

[0212] The wound treatment of the present disclosure provides definitive 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, freeze-dried fish skin grafts, are extremely effective in initiating and promoting the natural healing process. Skin substitutes, and especially physical scaffolds, and even more preferably fish skin physical scaffolds, allow cell infiltration and provide biomolecules that reduce inflammation and pain. These properties have been demonstrated multiple times in in vitro, in vivo, and clinical studies. In addition, in a further preferred embodiment, fish skin is naturally rich in omega-3, which has been shown to act as a barrier against bacterial invasion, with antiviral potential, bacteriostatic and antibacterial effects.

[0214] A preferred embodiment of fish skin provides bacterial barrier properties. Perhaps the most compelling evidence for the ability of unpigmented fish skin to reduce wound infection is an independent 21-patient study conducted at the Curie Institute in Paris, where infection rates for split-thickness donor sites dropped from 60% to 0% for wounds treated with fish skin. Even unpigmented 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, one of the MDR strains most frequently identified in combat trauma-related infections.

[0215] Methylene blue and gentian violet even provide additional antibacterial properties. Advances in wound treatment have resulted in the combination of antibacterial agents such as silver, iodine, and polyhexamethylene biguanide (PHMB) with traditional wound dressings. Although silver and iodine show robust effects in antibacterial activity, prolonged use of these agents results in high levels of cytotoxicity to host cells. MB and GV are FDA approved, can be used topically, and have demonstrated excellent efficacy for the management of chronic wounds with localized infection.

[0216] In vitro data shows promising results for the prototype of the preferred embodiment of the present disclosure. Testing was based on ASTM E2149 and Kirby-Bauer Zone of Inhibition assays. Both assays showed that fish skin explants impregnated with MB and GV efficiently inhibited both Escherichia coli (E. coli) or Staphylococcus aureus in solution and on agar plates.

[0217] Figures 14A and 14B show (A) the results of ASTM E2149 against E. coli, and Figure 14C shows (B) the results of Kirby-Bauer Zone of Inhibition assay against Staphylococcus aureus. Figures 14A and 14B show the results of the antibacterial fish skin placed in a suspension of E. coli and shaken for up to 24 hours, and a clear reduction in bacteria was observed between the antibacterial fish skin (disk 1) (Figure 14A) and the original fish skin (disk 2) (Figure 14B). Figure 14C shows that (B) fish skin treated with different concentrations of methylene blue and gentian violet ranging from 0.1% w / v (section 1410), 0.5% w / v (section 1420), and 1% w / v (section 1430) showed a clear zone of inhibition on the agar plate inoculated with Staphylococcus aureus, while the original fish skin showed no zone of inhibition.

[0218] Applicant has a wealth of scientific data demonstrating the healing properties of fish skin, including two randomized clinical trials on acute wounds in which fish skin was shown to provide more effective healing compared to mammalian cell and tissue-based products (CTPs), such as (Oasis)17 and human amniotic / chorion membranes with complete healing times, and clinical donor site studies in which the use of fish skin halved patient healing times. There are numerous independent published case series with overwhelmingly positive results regarding fish skin as an exemplary and preferred embodiment.

[0219] The creation of tissue regenerative wound treatments, and especially fish skin based tissue regenerative wound treatments, is highly feasible. The additional step of impregnating the skin substitute with antimicrobial color would require minimal addition of new equipment. MB and GV are readily available in pharmaceutical quality grades.

[0220] The tissue regeneration wound treatment embodiment of the present disclosure, and in particular the tissue regeneration wound treatment embodiment that includes fish skin as a skin substitute and is provided with antibacterial properties by either coloring agent(s) or additional active agents, can be effectively used as what can be called a temporary antibacterial scaffold for wound management, including diabetic foot ulcers, arterial ulcers, pressure ulcers, venous leg ulcers, and traumatic ulcers. The combination of these types of wounds is believed to be the cause of 54% of lower limb amputations in the United States, which is an irreversible debilitating condition. Nearly half of those who undergo amputation due to vascular disease die within five years. This is higher than the five-year mortality rate for breast, colon, and prostate cancer.

[0221] The standard of care in the United States for the treatment of chronic ulcers is as follows: The established usual or standard of care for chronic wounds incorporates common principles that apply to the management of all types of wounds, including: removing necrotic tissue by debridement (typically sharp debridement); maintaining fluid balance by selecting an appropriate wound dressing to control exudate; taking measures to prevent or treat wound infection; correcting ischemia in the wound area; for venous leg ulcers, applying some form of compression; and for diabetic foot ulcers, providing some form of offloading.

[0222] The tissue regeneration wound treatment embodiments of the present disclosure, and particularly those including fish skin as a skin substitute and provided with antimicrobial properties by either a colorant(s) or further additional active agents, may provide a more effective treatment of chronic wounds compared to the SOC established for skin substitutes, since the tissue regeneration wound treatment embodiments include fish skin as a skin substitute and are more effective as a treatment for chronic wounds by providing a temporary scaffold formation within the dressing to resist bacterial growth compared to the SOC. For the purposes of this application, it is expected that standard of care will be defined in the same manner as the Agency for Healthcare Research and Quality (AHRQ) definition. Predicate device decellularized fish skin wound products have been shown in randomized clinical trials to heal significantly faster compared to standard of care collagen dressings. The device provides a more effective treatment compared to the current standard of care as defined by AHRQ.

[0223] Embodiments of the tissue regeneration wound treatment of the present disclosure, and particularly those including fish skin as a skin substitute and provided with antibacterial properties either by colorant(s) or further additional active agents, achieve the same improvements compared to SOC while additionally providing resistance to bacterial growth and having distinct properties as a temporary scaffold.

[0224] A preferred embodiment of the tissue regeneration wound treatment of the present disclosure includes fish skin as a skin substitute, although of course other skin substitutes different from fish skin products or the fish skin-based wound treatment offered by Kerecis™ may be used.

[0225] In an extended comparison, the subject device provides a more effective treatment compared to emerging procedures, which include emerging procedures that are Q-coded as skin substitutes under the Healthcare Common Procedure Coding System (HCPCS).

[0226] As mentioned above, the group of skin substitutes that can be used as examples of skin substitutes according to the present disclosure is large and diverse. In the AHRQ technology assessment program entitled "Skin Substitutes for Treating Chronic Wounds" Technical Brief Project ID WNDT0818 published on February 2, 2020, in Table 2 on pages 9-13, 76 commercially available products are identified, but there are few studies comparing them in-house. Each of these listed skin substitutes can be an embodiment of a skin substitute according to the present disclosure.

[0227] The discussion of which treatments are more effective focuses on comparisons of treatment outcomes, antibacterial properties, temporary scaffold properties, and their impact on utilization.

[0228] The combination of antibacterial colors on a biodegradable scaffold should at least not interfere with the basic functions of each and may result in a synergistic additive effect.

[0229] Temporary scaffolding can be understood as a tissue scaffold that aids in tissue regeneration by supporting cell ingrowth, angiogenesis, and extracellular matrix regeneration. 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 adapted for use in wound care.

[0230] Antimicrobial products prevent bacterial colonization of the device but do not necessarily aid in scaffold formation (and in the case of silver-based products may actually be detrimental due to cytotoxic effects).

[0231] The tissue regeneration wound treatment of the present disclosure, and in particular the tissue regeneration wound treatment that includes fish skin as a skin substitute and is provided with antibacterial properties by either coloring agent(s) or additional active agents, provides what may be called "device identification." When an absorbent dressing is applied to a wound, it may be difficult to identify what is an active but partially absorbed device or what is wound slough tissue that should be removed. This may lead to premature dressing changes. Currently, there are no absorbent wound products that can identify colors.

[0232] The combination of scaffold and antibacterial color provides a synergistic additive effect.

[0233] The tissue regeneration wound treatment of the present disclosure, and in particular the tissue regeneration wound treatment comprising fish skin as a skin substitute and provided with antibacterial properties either by coloring agent(s) or by further additional active agents, is the first wound care product known to the inventors that provides temporary scaffolding and device identification of absorbent wound dressing. Combined with antibacterial protection, it limits the risk of bacteria causing inflammation or growing in the product and promoting degradation. Furthermore, easy device identification allows for more accurate dressing changes.

[0234] Temporary scaffolding can be compared to other skin substitutes. Temporary scaffolding supports cell ingrowth, vascularization, and extracellular matrix regeneration. As the field of tissue engineering continues to evolve, the criteria for an ideal skin graft have shifted to a material that supports cell integration and tissue growth. These criteria include that the scaffold must meet one or more, preferably all of the following: allow and promote cell ingrowth; allow uniform spatial distribution of cells; support extracellular matrix regeneration; support vascularization; do not cause a foreign body type reaction; rapidly integrate into the wound; and be mechanically strong and stable.

[0235] The inventors have shown that the fish skin implantation technique as described in this disclosure can provide a temporary scaffold function, and furthermore, evidence shows that the results of the scaffold formation for cell ingrowth, vascularization, and extracellular matrix regeneration are more effective than other known devices, such as absorbable collagen devices, e.g., Primatrix.

[0236] Based on these results, evidence exists that the tissue regeneration wound treatments disclosed herein, and in particular those including fish skin as a skin substitute and provided with antimicrobial properties either by colorant(s) or further additional active agents, provide a more effective treatment than standard of care by functioning as a temporary scaffold.

[0237] The addition of antimicrobial agents to the original fish skin provides antimicrobial protection to the device. It has also been discovered by the inventors that the addition of suitable coloring agents does not interfere with the basic scaffolding effect of the fish skin. MB and GV are organic dyes that can be used to reduce microorganisms in clinical settings with minimal toxicity to humans. MB and GV can be used topically for rapid management of localized bacterial load within the wound. The concentration of MB and GV in the preferred embodiment is controlled to 0.00025 g / g (0.01%) or less, which is lower than that of Hydrofera Blue Ready (0.0035 g / g or less for each color) and significantly lower than the concentration of MB and GV in the commercially available topical agent 1%. Of course, Hydrofera Blue Ready may be used as another embodiment of the coloring agent. GV and MB can be used in conjunction with enzymatic debridement agents, growth factors, and hydrogels without inhibiting the action of the companion products.

[0238] The inventors have discovered that the MB and GV used in the tissue regeneration wound treatment disclosed herein, particularly the tissue regeneration wound treatment involving fish skin, do not impair the scaffolding effect of fish skin. The addition of MB and GV to fish skin can be performed at the final stage of the manufacturing process before sterilization. In this process, the design, material, function, packaging, and sterilization of the original fish skin are not changed.

[0239] A recent study (Stone II, International Journal of Molecular Sciences, 2021) compared fish skin grafts to fetal bovine dermis (Primatrix) in treating deep partial thickness (DPT) burns in a preclinical porcine model. The aim of the study was to determine how effective fish skin grafts are in treating DPT burns, how they integrate, and whether they improve long-term healing. Under the conditions of the study, the fish skin grafts were found to integrate into the wound bed faster than fetal bovine dermis. The fish skin grafts resulted in faster re-epithelialization starting at day 10 and by day 28, especially at day 14, with the differences between the fish skin grafts and fetal bovine dermis being notable. The fish skin grafts resulted in increased blood flow and newly formed blood vessels. The fish skin grafts promoted full formation of the epidermis after 21 days. The fish skin grafts also caused less of an inflammatory response (less foreign material and fewer inflammatory cells).

[0240] While the results of this study provide evidence that fish skin grafts are the preferred embodiment, fetal bovine dermis (Primatrix) products may, of course, still be used as an effective skin substitute in accordance with the present disclosure, and under some conditions or considerations may also be the preferred embodiment of a skin substitute as contemplated in the present disclosure.

[0241] Furthermore, although this study was performed with an unpigmented version of the fish skin product without the antimicrobial agents MB and GV, the extent of the wounds created 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 the fish skin in this study is to act not only as a temporary covering but also as a temporary scaffold for long-term healing. This study provides a number of important insights into the scaffolding effect of intact fish skin.

[0242] The tissue regeneration wound treatments of the present disclosure, and in particular those including fish skin as a skin substitute and provided with antimicrobial properties either by colorant(s) or further additional active agents, prevent bacterial colonization of the subject devices compared to other currently known skin substitutes.

[0243] The phrase "bacterial barrier" may be understood to mean that the broad-spectrum antimicrobial agent provides a barrier against bacterial invasion of the dressing, which may help reduce infection and ensure that the temporary scaffold functions as intended.

[0244] Skin substitutes in the broad sense can be considered as biodegradable tissues that are infiltrated by the body's own cells and then integrated, absorbed, or degraded. Most skin substitutes have a low innate ability to defend against bacterial invasion and can become colonized if bacteria are present in the wound. Bacterial colonization of skin substitutes results in more rapid degradation and less ingrowth of host cells.

[0245] Among the 76 skin substitutes, two other skin substitutes that provide some antibacterial effects are listed, namely PriMatrix AG and PuraplyAM. However, neither of these two were found to have the same antibacterial spectrum and activity as the antibacterial agent used in the tissue regeneration wound treatment of the present disclosure, and in particular the tissue regeneration wound treatment that includes fish skin as a skin substitute and is provided with antibacterial properties by either a coloring agent(s) or a further additional active agent. Of course, as mentioned above, PriMatrix AG and PuraplyAM may still be considered as embodiments of the skin substitute in the present disclosure, and may in fact be preferred embodiments under certain circumstances and conditions.

[0246] [Table 2] TIFF2024510837000008.tif246163

[0247] Of course, as noted above, PriMatrix AG and Puraply AM may still be considered embodiments of the skin substitutes in this disclosure, and may, in fact, be preferred embodiments under certain circumstances and conditions. The antimicrobial range of the subject devices is comparable to Hydrofera Blue, a dressing with comparable MB and GV concentrations.

[0248] The tissue regeneration wound treatments of the present disclosure, and particularly those including fish skin as a skin substitute and provided with antimicrobial properties either by coloring agent(s) or further additional active agents, may provide what may be the term "device identification" that promotes optimal utilization cycles. "Device identification" may be understood as a coloring agent that facilitates identification of the product when integrated into the wound bed.

[0249] Skin substitutes are most transparent or off-white prior to application and become clear, white, or caramel-colored upon integration into the wound bed. This appearance, especially for inexperienced users, is sometimes indistinguishable from wound slough tissue, exudate, or biofilm. This makes it difficult to determine whether the skin substitute is fully integrated and in need of replacement, or whether it is still partially active and can be kept in the wound longer. The inability to determine whether there is still active skin substitute in the wound can lead to three outcomes: (1) misidentifying the slough tissue in the wound as a collagen dressing, leading to the healthcare provider not removing the slough tissue from the wound, thus slowing wound healing and increasing the risk of infection; (2) misidentifying the active products in the wound as slough tissue, leading to the healthcare provider prematurely removing the device; and (3) removal of the temporary active scaffold tissue with the ingrowth of fresh host cells.

[0250] The active products within the wound are mistaken for sloughed tissue, leading to premature reapplication of the device with unnecessary intervention and associated costs for the patient.

[0251] The novel devices as disclosed herein are colored using biocompatible colorants that allow safe and easy differentiation from sloughed or other tissues. This is done using the disclosed colorants that bond color to the implant.

[0252] The device represents a breakthrough technology and a novel application of an innovation that may lead to clinical improvements in the treatment of chronic non-healing wounds and potential prevention of amputation. The device provides a 3D structure that supports human cell infiltration and proliferation, and vascularization, while inhibiting bacterial colonization on the scaffold.

[0253] The tissue regeneration wound treatments of the present disclosure, and in particular those including fish skin as a skin substitute and provided with antimicrobial properties either by colorant(s) or further additional active agents, have at least one or more, and preferably all of the following characteristics: provide a stable, absorbent scaffold that promotes cellular ingrowth and vascularization; provide broad spectrum coverage to address microorganisms often present within the wound; do not cause toxicity to host cells and inhibit cellular ingrowth compared to silver-containing dressings; do not cause mutations in changes in bacterial antimicrobial resistance compared to antimicrobial dressings.

[0254] Additionally, depletion of the colorant may result in a change in color of the dressing, which may provide an important visual indicator to guide dressing changes.

[0255] Applicants have multiple in vitro and in vivo temporary scaffolding data for the Omega3 Wound, a previously defined device. Our evidence shows that the addition of a colorant (antimicrobial agent) to the scaffold does not interfere with basic function and has a synergistic additive effect.

[0256] In vitro studies of cell ingrowth (Magnusson, Military Medicine, 2017) found that fibroblasts infiltrated and remodeled fish skin grafts after 12 days compared to hHACM materials with less fibroblast infiltration. The tissue regeneration wound treatments disclosed herein, and in particular those including fish skin as a skin substitute and provided with antibacterial properties by either colorant(s) or further additional active agents, retain the same porous structure and pore size as the original fish skin, which attracts 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 raise concerns of cytotoxicity. Furthermore, since the reference device, Hydrofera Blue Lady, contained higher concentrations of MB and GV but did not raise concerns of cytotoxicity, the tissue regeneration wound treatment of the present disclosure, and in particular tissue regeneration wound treatments including fish skin as a skin substitute and provided with antimicrobial properties either by colorant(s) or further additional active agents, should not cause any adverse effects on cellular ingrowth.

[0257] Preliminary bench studies have demonstrated the efficacy of the disclosed tissue regeneration wound treatments, and in particular the tissue regeneration wound treatments containing fish skin as a skin substitute and provided with antibacterial properties by either one or more of the colorants (MB and GV), on antibacterial potential. The tests were performed using three microorganisms most commonly found in wound infections: E. coli, S. aureus, and P. aeruginosa. Testing methods ranged from simple and basic assays such as agar disk diffusion to more challenging industry standard tests such as AATCC100 and ASTM E2149. The agar disk diffusion results showed that compared to Omega3 Wound and Primatrix AG, the tissue regeneration wound treatments of the disclosed tissue regeneration wound treatments containing fish skin as a skin substitute and provided with antibacterial properties by either one or more of the colorants (MB and GV) showed the formation of a clear zone of inhibition against S. aureus. The diameter of the inhibition zone was 17.25+0.5mm for the tissue regeneration wound treatment of the present disclosure, which included fish skin as a skin substitute and was provided with antibacterial properties by one or more colorants (MB and GV), and 11.67+0.58mm for Primatrix AG, where fish skin showed no effect, and thus had an inhibition zone of the same diameter as the sample diameter (6mm). The results of the AATCC100 evaluation demonstrated the high efficacy of the tissue regeneration wound treatment of the present disclosure, which included fish skin as a skin substitute and was provided with antibacterial properties by one or more colorants (MB and GV), on Staphylococcus aureus and Pseudomonas aeruginosa. The reduction rate was estimated to be approximately 98% for Pseudomonas aeruginosa. The tissue regeneration wound treatment of the present disclosure, which included fish skin as a skin substitute and was provided with antibacterial properties by one or more colorants (MB and GV), showed strong antibacterial efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa. Results of ASTM E2149 testing demonstrated reduced growth in E. coli suspension with Kroma Antivirus: 37 colonies formed on agar plates using the tissue regeneration wound treatment of the present disclosure, which included fish skin as a skin substitute and was provided with antimicrobial properties by one of the coloring agents (MB and GV), while there were 445 and 491 colonies on the uncolored fish skin and E. coli suspension by itself.Benefiting from the disclosed tissue regenerative wound treatment, which included fish skin as a skin substitute and was provided with antibacterial properties by one of the coloring agents (MB and GV), the proliferation reduction rate was approximately 92-93%.

[0258] Given our promising results from our preliminary testing, the disclosed tissue regeneration wound treatment including fish skin as a skin substitute and provided with antibacterial properties by one of the coloring agent(s) (MB and GV) will provide 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 their EMC template-like properties enhance cell ingrowth and tissue regeneration.

[0260] The inventors have discovered and disclosed various embodiments, including a preferred embodiment that uses a fish skin temporary scaffold in combination with two antimicrobial colorants for wound management as an effective barrier to resist microbial colonization within the scaffold. The temporary scaffold supports angiogenesis and cellular ingrowth while inhibiting microbial colonization at the dressing.

[0261] The tissue regeneration wound treatment of the preferred embodiment of the present disclosure is an acellular absorbable fish skin wound matrix, which includes fish skin as a skin substitute and is provided with antimicrobial properties by one of the coloring agents (MB and GV). The wound treatment acts as a temporary scaffold that supports angiogenesis and cellular ingrowth while inhibiting bacterial colonization on the scaffold. The device includes two antimicrobial agents that provide broad-spectrum antimicrobial protection on the scaffold with methylene blue and gentian violet (crystal violet). The subject device is supplied as a sterile, intact or mesh sheet with a size of up to 20 x 30 cm. The broad-spectrum antimicrobial agent provides a barrier against bacterial ingress of the dressing, which may help reduce infection and ensure that the temporary scaffold functions as intended.

[0262] Indications for use The tissue regeneration wound treatment of the preferred embodiments 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] Configuring Devices The tissue regeneration wound treatment of the preferred embodiment is a fish skin medical device for wound management. The subject scaffold material, hereinafter sometimes referred to as the device, is obtained from the skin of wild North Atlantic cod (Gadus morhua) by a standardized and controlled manufacturing process and is supplied in terminally sterile packaging of skin pouches in the following sizes: 16mm disk; 2x2cm; 2x4cm; 5x5cm; 10x10cm; 20x30cm. The device may also be provided in a granulated form, as described and shown above.

[0264] The device may contain two antimicrobial agents, such as methylene blue and gentian violet (crystal violet), which provide broad-spectrum antimicrobial protection on the scaffold. The concentrations of MB and GV are controlled to below 0.00025 g / g (0.01%), but may include up to 0.1%.

[0265] The subject devices preferably fully integrate into the surrounding tissue over time in response to the deposition of new host tissue. The favorable physical properties of the subject devices allow for cellular ingrowth. The subject devices are preferably biocompatible, non-crosslinked, bioabsorbable, strong, and flexible. Their tensile strength supports fixation with sutures or staples.

[0266] The mechanism of action of the subject device can be broken down into three main areas: 1. Collagen Dressing: Substantially equivalent to a decellularized fish skin wound product (K132343) with the following differentiated properties: 1.1. Impregnated with antimicrobial colorants: 2a: "Bacterial Barrier": The broad spectrum antimicrobial provides a barrier against bacterial intrusion of the dressing to help mitigate infection and ensure that the temporary scaffold functions as intended. 2b: "Device Identification": The colorants facilitate identification of the product when integrating into the wound bed. 2. "Temporary Scaffold Formation": Temporary scaffold formation that supports cellular ingrowth, vascularization, and extracellular matrix regeneration 2.1. Collagen Dressing. The subject device functions substantially equivalent to a decellularized fish skin wound product as a collagen dressing with the same basic mechanism of action.

[0267] The main purpose of collagen scaffolds is to act as a template that mimics the extracellular matrix (EMC) of healthy tissue. By mimicking and supporting cells, it aids in the remodeling of many different types of tissues, aiding the wound healing process. Each component of the EMC is essential for each of the stages of wound healing. Components of the ECM play a key role in aiding cell proliferation and differentiation, directing cell migration, and regulating cellular responses. Exogenous EMC undergoes natural remodeling of healthy tissue at the wound site, being degraded and replaced by native collagen. Decellularized fish skin wound products re-establish functional EMC in chronic wounds. Collagen dressings provide: (1) a moist wound environment, (2) fluid management, and (3) controlled fluid transpiration.

[0268] The decellularized fish skin wound product is substantially equivalent to many of the porcine collagen matrices approved through the 510k premarket notification process and may be used as a collagen dressing. The effectiveness of the device as a collagen dressing for wound management was demonstrated in a non-inferiority study compared to Oasis Wound Matrix, a mammalian-derived collagen dressing. The study concluded that the fish collagen dressing was non-inferior to the porcine-derived collagen dressing, did not identify any side effects, and showed improved wound healing over a 28-week period.

[0269] The only technical difference between the decellularized fish skin wound product and the subject device is the addition of coloring agents. There is no evidence or literature that any coloring agents destroy or crosslink the collagen scaffold. Therefore, based on our evidence, there is sufficient evidence to suggest that the subject device, whether it is a fish skin-based skin substitute or other skin substitute, also serves as a scaffold that mimics the extracellular matrix to support cell ingrowth and vascularization.

[0270] The stains of the preferred embodiments have significant antimicrobial effects. Using a stain 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. Upon contact with bacteria, the MB and GV in the subject device eliminate the bacteria by making bacterial growth unsustainable within the device.

[0271] MB is part of the phenothiazine family and was one of the first FDA-approved treatments for malaria when resistance to antimalarial drugs emerged. MB has demonstrated bacterial inactivation in vitro with a wide range of microorganisms, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.

[0272] The MB and GV dyes act as indicators to distinguish between the device and slough tissue in the wound. Because the skin substitute is an absorbent dressing, the color notifies the clinician when the dressing is fully integrated and a second application is required. The color also helps the clinician reduce accidental dressing removal when performing debridement on the wound. Unpigmented collagen dressings can sometimes be difficult to distinguish from slough tissue when partially integrated.

[0273] For example, FIG. 15A shows a wound graft that has turned into slough in a wound filled with bacteria. In comparison, FIG. 15B shows a wound graft of fish skin that is approximately 50% integrated and should remain in the wound. However, as can be seen by comparing the wound slough in FIG. 15A with the wound graft in FIG. 15B, it is difficult to accurately and easily distinguish between grafts that have undergone ingrowth and those that have become slough. In comparison, FIG. 15C shows a skin substitute, in this case a fish skin graft, stained with MB / GV stain. This is evident from the fact that the graft in FIG. 15C has integrated and ingrowth has occurred, and that the graft in FIG. 15C should remain for another week and should not be removed.

[0274] In vitro studies with mouse fetal fibroblasts added to the top of fish skin have shown 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 faster wound healing and showed an increase in newly formed blood vessels, in addition to superior blood flow under the fish skin. In the same pig study, fish skin grafts promoted complete formation of the epidermis after 21 days, resulting in faster re-epithelialization and less inflammatory response.

[0275] When applied to a patient's wound, embodiments of the subject device rapidly integrate into the wound and provide a temporary scaffold for cell migration and proliferation, while MB and GV molecules inhibit and eliminate microbial colonization on the matrix. Abundant dermal collagen fibers support cell ingrowth, angiogenesis, and extracellular matrix regeneration, which are important for speeding wound healing.

[0276] Pigmented skin substitutes, such as fish skin grafts, are eventually degraded in the body. Cellular ingrowth of primary fibroblasts, eventually with some inflammatory components, completely remodels and degrades the original skin substitute, such as a fish skin graft, and the color.

[0277] The enzymatic process is primarily the hydrolysis of collagen into smaller, easier to process particles and the reduction of colorants.

[0278] Pigmented skin substitutes, such as fish skin grafts, may have dimensions of up to 7×20 cm, or even 20×40 cm, may be solid or meshed, and may be in sheet or particulate form.

[0279] Further Examples of Production of Wound Treatments In yet another example of a method or procedure for the production of an embodiment of a tissue regeneration wound treatment, the following procedure was followed.

[0280] Ten skins had been packed and frozen flat prior to our arrival.

[0281] A colour solution containing dilutions of 0.01% and 0.005% MB&GV was prepared by mixing.

[0282] To obtain clean water, a sink was used to monitor bacterial load and was boiled before use.

[0283] First, a 1% stock solution was made.

[0284] GV: 650mg Pharmaceutical Grade (USP), SA-1290002, LOT G1K417, SP1098511 (Distica)

[0285] MB: Methylene Blue hydrated for microscopy, ≥97%.0%, Sigma-Aldrich 66720-100g, LOT #BCBZ4929

[0286] 0.4g MB + 0.4g GV was added to 40ml 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). From one bottle, 10ml was removed with a clean pipette and from the other bottle, 20ml of water was removed. These volumes were replaced with stock solutions to make 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, meaning that vigorous cleaning of all surfaces with water and ethanol was required.

[0289] The fish skins were then removed from the freezer, placed on ice and transported with the staining solution. All were brought into the high-risk area chamber next to the freeze dryer. The fish skins were thawed under running tap water from the tap in the high-risk chamber. As the fish skins were quite large and long, once they were soft and pliable they were cut into two shorter pieces. Five fish skins (10 equal pieces) were placed in two aluminum trays for staining.

[0290] Approximately 700 ml of Kroma solution was placed into each tray, labeled 0.01% and 0.005%, respectively. The two trays were then placed into a plastic bag, folded to reduce the risk of spillage, and placed on a shaker set at 30 rpm for 2 hours.

[0291] After approximately 20-30 minutes, the fish skins were moved with sterile wipes to encourage even dyeing. After approximately 20 minutes, it was apparent that the dye solution had reduced its density and become transparent as the fish skin absorbed the dye. To compensate for this, approximately 300ml of dye solution was added to each tray, meaning that the final dye volume was approximately 1000ml.

[0292] A portion of both the original coloring solution and the remainder of the used staining solution is saved in a 50 ml tube to allow for later quantification of the concentration. This, combined with measurements of the size and weight of the fish skin after freeze-drying, allowed a rough quantification of the absorption of the color into the fish skin.

[0293] The freeze dryer was started just after 18:00 as it takes about 45 minutes to be ready to run.

[0294] Due to a computer error, the freeze dryer had difficulty starting up, so the staining took approximately 3 hours (note that the shaker reverts to the faster default shaking speed after 3 hours). The skin was washed thoroughly with running tap water for 10-15 minutes in a high-risk room.

[0295] The remaining staining solution was apparently somewhat clearer again, with slight color differences in the two batches of fish skin, with the 0.01% being a true denim dark blue, while the 0.005% was rather 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 from both batches to have a total of three complete plates. The more heavily stained skin was on the left hand side of the shared plate.

[0296] Freeze drying was started at approximately 8:30 pm and left overnight.

[0297] The fish skins were freeze-dried in the morning and packaged for non-sterile use. Another batch was repeated the next day using the remaining 0.01 and 0.005% solutions (at room temperature).

[0298] Further Examples of Prototype Wound Treatment Generation Two prototypes of colored cod skin are generated with two different concentrations of methylin blue and gentian violet: 0.01% w / v in water; methylin blue (50%) and gentian violet (50%), and 0.005% w / v in water; methylin 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 w / v; methylin blue (50%) and gentian violet (50%); 1 liter of 0.005% w / v aqueous solution w / v; methylin blue (50%) and gentian violet (50%); 10 aluminum trays; scissors; small Tyvek pouch; large Tyvek pouch; large plastic bag; shaker; sealer.

[0300] Prototype process: All cod skins were fresh from production on the same day. Frozen for 5 hours at -80°C. As the cod skins were too large to fit into the aluminum trays, they were cut into two pieces, resulting in a total of 20 cod skins.

[0301] Prototype 0.01% One litre of the 0.01% solution was poured into an aluminium tray marked MB-GV 0.01% and 10 cod were placed evenly onto the tray, ensuring the solution covered the skin. The tray was placed into a plastic bag to minimise the risk of colour spilling off and the tray was then placed on a shaker at pro:40 for 3 hours.

[0302] Prototype 0.005% One litre of the 0.005% solution was poured into an aluminium tray marked MB-GV 0.005% and 10 cod were placed evenly onto the tray, ensuring the solution covered the skin. The tray was placed into a plastic bag to minimise the risk of colour spilling off and the tray was then placed on a shaker at pro:40 for 3.5 hours.

[0303] Start of coloring on shaker: 15:40 ± 10 min

[0304] End of coloring on shaker: 19:05 ± 5 minutes

[0305] Rinse start time: 19:05 ± 5 minutes

[0306] Rinse end 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 dryer program: SvavaColor - 10 hours total time.

[0308] Packaging: Clean, dry, pigmented cod skin was supported and prepared for packaging by visual inspection and bend test. Samples were packaged into Tyvek pouches, packaged into small and large sample pouches, marked and sealed.

[0309] No skin scraping was performed on these prototypes.

[0310] Crosslinking to improve color fastness and mechanical properties of wound treatments In further embodiments, it has been discovered by the inventors that crosslinking the skin substitute, e.g., scaffold material, can further enhance the properties of the skin substitute, including increasing the fastness of colorants that color the skin substitute, increasing the mechanical material properties of the skin substitute, increasing the resistance to enzymatic and chemical degradation in the skin substitute, and extending the lifespan of colorants added to the skin substitute under biological conditions such as a treated wound. In a preferred embodiment, the primary purpose of crosslinking the skin substitute, e.g., scaffold material, is to obtain a colored product that maintains its color for at least 1 day after application to the wound, preferably 3 days after application to the wound, and more preferably up to 8-10 days, and even more preferably up to 14 days after application to the wound.

[0311] As described herein, crosslinking of the skin substitute and / or the skin substitute loaded with colorants can be accomplished by a variety of means, for example, by irradiation or chemical means.

[0312] Chemical crosslinks or modifiers In one embodiment, the skin substitute is crosslinked by treating the skin substitute with a crosslinking agent, hi another embodiment, the proteins of the skin substitute are otherwise modified by treating the skin substitute with a protein modifying agent.

[0313] In embodiments, the chemical crosslinkers target one or more of the following groups: primary amines (-NH2); carboxyls (-COOH); sulfhydryls (-SH) or carbonyls (-CHO), or other groups. Thus, crosslinkers can be, for example, amine-reactive, carboxyl-to-amine-reactive, sulfhydryl-reactive, and / or aldehyde-reactive.

[0314] In a first embodiment, the cross-linker is a simple sugar or monosaccharide. Alternative sugars may be used, including glucose, fructose, and galactose. For example, the cross-linker may be or include ribose. Alternative sugars may be used, including glucose, fructose, and galactose. Oligosaccharides and disaccharides may also be considered.

[0315] In another embodiment, the crosslinker is a natural or synthetic crosslinker, for example, in an embodiment, the crosslinker includes or is genipin.

[0316] Example 1 - Ribose cross-linking A first example is provided herein in which ribose is used as a cross-linker.

[0317] According to this example, a standard ribose (stock) solution is prepared. For this, a 0.2M (molar) solution of ribose is made in PBS containing 0.05% (w / v) sodium azide to prevent bacterial growth. Other concentrations of ribose may be used, and other bacterial growth inhibitors may be used. In this example, the solution contains, by weight, 30.03 g of ribose, 9.55 g of premixed 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 to 1.00 L with deionized water. The components are mixed until completely dissolved, after which the solution is ready for use.

[0318] In this example, a Kerecis™ fish skin derived cell scaffold product is used as a skin substitute. In general, any size and / or number of scaffolds could be processed, even including particulated scaffold material. The container in which the scaffold material is added to the solution can be large, and the volume of the ribose solution completely covers the sample. In this example, the scaffold material is placed in a 4×8 cm 2The cod was cut into pieces of 1.5 mm each. Five pieces or samples were cut from the larger sample with the long side (8 cm) parallel to the length of the cod skin. The samples were then soaked in about 250 mL of ribose solution at room temperature for 3-6 days. The first sample was removed at the 3 day mark, the next two at the 5th day and the last two at the 6th day. Additional samples of the same size were also prepared by soaking in about 80 mL for 40 hours.

[0319] After removal from the ribose solution, each sample was washed with running water and then placed in a water bath for 2 days to wash away unreacted ribose along with PBS and sodium azide. The water was further changed periodically (once or twice daily) to aid in the washing process. The samples were then partially dried and frozen for further processing.

[0320] Staining methods for ribose cross-linked scaffolds: Two common methods have been used to stain cross-linked scaffolds. One can be described as meta-staining, where MB and GV are added to the cross-linking solution. In this method, one 4 × 8 cm 2 The scaffold pieces were soaked 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, the stock solutions being 0.1% by weight, resulting in a concentration of MB / GV in the solution of 0.002%. After the combined crosslinking / staining process, in the same manner as described above for crosslinking, the samples were first washed with tap water and then left in water for 2 days to produce sample 1610 as shown in FIG. 16. Sample 1610 in FIG. 16 is a meta-stained, ribose-crosslinked, stained scaffold that was left in the "meta" solution for 24 hours.

[0321] According to the second method, the post-staining used the same conditions as those previously discussed for the "standard staining process", i.e., after the scaffolds had undergone the cross-linking and washing process, the samples were stained in a 0.002 wt% solution of MB / GV in PBS for 3 h, for which a 4 × 4 cm 2100 mL of scaffold pieces were stained in 100 mL of solution. This can be done with scaffolds that were pre-crosslinked regardless of crosslinking time, e.g., 24 hours, 40 hours, 5 days, or 6 days. Figure 17 shows a post-stained, ribose-crosslinked scaffold 1710 that 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 variations from the ribose crosslinking example above. The process of meta-staining can be modified in at least two ways. The first modification may include increasing or decreasing the time in solution. The second modification may include changing the concentration of either the dye or ribose in the solution. Since the absorption of the dye occurs relatively slowly over time and is directly related to the dye concentration in the solution, for example, if the meta-staining is for 48 hours, it may be productive to lower the concentration of MB and GV in the solution if the concentration of color in the scaffold needs to be the same as in the 24-hour process described above, and in fact any combination of time and concentration (within reason) is possible and will produce unique results.

[0323] For post-dyeing as described above, the crosslinking time of the scaffold can be varied. If changes in the concentration of dye within the scaffold are effected, the concentration of dye and / or time in the dye solution can also be altered.

[0324] Example 2 - Genipin Crosslinking In the second example, genipin is used as the cross-linking agent, as described above, and an exemplary procedure for this is now described.

[0325] Prepare genipin crosslinking solution. According to this example, a 0.3% (w / v) solution of genipin in PBS was made by dissolving 0.60 g of genipin in 200 mL of pre-made PBS solution (9.55 g of pre-made PBS powder / 1 L) to make 200 mL of solution. Stir the solution until no solid particles remain.

[0326] In this example, Kerecis™ fish skin derived cell scaffold product is used again as a skin substitute. In general, any size and / or number of scaffolds could be processed, even including particulated scaffold materials. In this example, a culture plate with 15 mL wells was used. 2×2 cm 2 scaffold / collagen pieces were placed in each well and genipin solution was added. Each well was completely filled with a total of 15 mL of solution. The plate was then immersed in a 37°C water bath for 24 hours after closing and sealing with a lid. Note that if the temperature is constant at 37°C, any other heating source can work and evaporation is limited by sealing the container or recondensing the solution. After 24 hours in the solution, the scaffolds were washed with water and frozen. In this example, crosslinking with genipin caused the scaffold material to roll up, as well as turning the scaffolds bluish black. There was also a clear difference in the stiffness of the samples.

[0327] Similar to ribose cross-linking and staining, the two methods explored in this example are staining during and after cross-linking, i.e., meta-staining and post-staining. In this example, six wells were used, four of which contained only 15 mL of 0.3% genipin solution and two of which contained MB and GV as well (meta-staining). The meta-staining solution was prepared by adding 150 μL of each dye stock solution (0.1 wt%) to the wells, followed by the addition of 14.7 mL of genipin solution. With the exception of the addition of MB / GV, all six wells were identical and underwent the same treatment during the cross-linking process.

[0328] The post-staining procedure for genipin cross-linked scaffolds is the same as for ribose cross-linked scaffolds. Samples are immersed in 0.002% MB / GV in PBS solution for 3 h and stained on 2 × 2 cm 2 Now, 50 mL of solution is used to cut two 2 x 2 cm cubes to produce sample 1810 in Figure 18. 218 shows a post-stained genipin scaffold, sample 1810, stained in 0.002 wt% MB / GV in PBS for 3 hours. After staining, the samples were washed, partially dried, and frozen.

[0329] Other embodiments and exemplary methods may include variations from the above genipin cross-linking example. The modifications that can be made to the staining process of genipin cross-linked scaffold samples are essentially the same as for the ribose method described above. That is, regardless of the process (meta or post-staining), the time and concentration of the dye can be changed.

[0330] Figures 19A and 19B show a comparison of the improvement of color retention by chemical cross-linking. 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 a Kerecis™ fish skin-derived cell scaffold product that had been left in 0.002% MB / GV, PBS solution for 3 hours. The sample from which fragment 19-C was taken was also cross-linked with 0.3% genipin solution according to Example 2 above. The sample from which fragment 19-B was taken was also cross-linked with ribose solution according to Example 1 above. Additionally, the sample from which fragment 19-A was taken was not cross-linked, but only colored in 0.002% MB / GV, PBS solution for 3 hours.

[0331] For comparison, FIG. 19A shows fragments 19-C, 19-B, and 19-A, and samples for 19-C and 19-B, respectively, in dishes 1930, 1920, and 1910 after staining. Subsequently, a bicarbonate solution of pH 8 was added in equal volume and concentration to each of dishes 1930, 1920, and 1910. Fragments 19-C, 19-B, and 19-A were stored at a temperature of 37° C. in dishes 1930, 1920, and 1910, respectively, for 48 hours, resulting in the same fragments 19-C, 19-B, and 19-A after 48 hours, as shown in FIG. 19B.

[0332] As can be seen, the color fastness of fragments 19-C and 19-B, cross-linked with genipin (19-C) and ribose (19-B), respectively, was significantly improved over the similarly colored but uncross-linked 19-A. The cross-linked pieces 19-C and 19-B clearly show that their coloration 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 cross-linking process can be modified as well. This affects the final color for both procedures in the case of meta-staining, but has a greater impact in the case of genipin, since the resulting color obtained directly from cross-linking when using the above method is very dark and concentrated. If the time, concentration, or temperature in the solution is reduced, the result will be less cross-linking and a lighter color, as shown in several studies.

[0334] Radiation crosslinking In another embodiment, the skin substitute is crosslinked by irradiating the skin substitute material with electromagnetic radiation. In a first example, the skin substitute, for example a scaffold 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 mL of sterile water to 200 mg of MB and stirring until the color dissolved. A PBS solution was also prepared by mixing 1 liter of liquid 10x PBS with 8.8 liters of tap water and stirring.

[0336] Kerecis™ fish skin derived cell scaffold product was used again as skin substitute. In general, any size and / or number of scaffolds can be processed, even including granulated scaffold material, which can have a diameter as small as 1 mm. In this example, the pieces used included 14 pieces of fish skin cut to 4×8 cm and 2 pieces of uncut fish skin. All fish skin was previously scraped to remove flesh tissue, scales, and fascia.

[0337] The PBS and a portion of the stock solution were mixed in a large container until homogenous. The volume of each solution was 8.8 L for PBS stock and 200 mL for stock color.

[0338] After the color solution was prepared, the fish skins were added to the color solution and stirred to ensure that the fish skins did not stick together.

[0339] The fish skins were left in the colour solution for 3.5 hours, stirring every hour. A UV cabinet was set up and the fish skins were arranged on a tray.

[0340] The UV radiation source was a 15W UV light (254 nm) that was screwed to 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 redirected to face the fish skin. The tray was positioned approximately 12 cm away from the light. In this example, a UV light was selected that had nearly monochromatic UV radiation, but in other embodiments, other UV sources of various wattages and wavelengths may be used, either monochromatic or polychromatic UV radiation, with the UV radiation having a wavelength in the range of about 10 nm to about 400 nm.

[0341] Thus, samples were obtained including: Sample A, which was removed from the MB-based color solution, placed in a PBS solution (without color) and exposed to UV radiation; Sample B, which was placed in the MB color solution and exposed to UV radiation while in the MB color solution; and Sample C, which was placed in the color solution but not exposed to UV radiation. In other words, the skin of Sample A can be considered similar to post-dyeing crosslinking in that the skin of Sample A was dyed in the MB-based color solution and then crosslinked by UV radiation only while in the PBS solution. In comparison, the skin of Sample B can be considered similar to meta-dyeing in that the skin of Sample B remained in the MB-based color solution while being crosslinked by UV radiation. Also, the skin of Sample C can be considered a control in that the skin of Sample C was dyed in the MB-based color solution but was not exposed to UV radiation during or after dyeing. However, as a control, the skin of Sample C was kept in a UV cabinet but in a dark part of the cabinet where it was not exposed to UV radiation. Thus, the skin of Sample C was treated with similar temperature, flipping, and time conditions for comparison to the crosslinked skins of Samples A and B.

[0342] The skins were left in their respective liquid solutions for 6 hours. Because the skins were floating on top, they were flipped upside down to ensure that both sides were evenly exposed to the UV light. This was also done for the skin in the tray that had not been exposed to the UV light (in the dark), which went through the same process as the skin under the UV light. When the skins were flipped 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 over, and as 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 an extent that would require a cooling system.

[0344] After the radiation step, the skin was collected from the tray and transferred into three separate bags, one each for Sample A, Sample B, and Sample C. The skin was rinsed in cold water for a few minutes and placed on a steel plate before being inserted into the freeze dryer. The skin was left in the freeze dryer overnight.

[0345] The skin was collected into three separate bags and each piece was sealed in a Tyvek bag.

[0346] Some pieces of the various samples were sterilized using ethylene oxide.

[0347] Sample A: The skin of Sample A was stained in MB color solution, then removed therefrom, rinsed, and placed in PBS solution under UV light. The PBS solution had no color originally, but at the end of the UV radiation, it was clear that the color from the skin from the previous staining had leaked out of the skin and stained the PBS solution. The final color of the skin was a lighter blue and even a little green on the skin compared to the other prototypes, which were a darker blue.

[0348] Samples B and C: Comparing fragment 20-B of Sample B with fragment 20-C of Sample C, as seen in FIG. 20A, once crosslinking of the skin of Sample B was completed, there was no visible difference between the pigmented skin of Sample B that was placed under UV light while in the MB-based color solution (Sample B) compared to the skin of Sample C that was exposed to UV light. For comparison, fragment 20-A of Sample A is also provided in FIG. 20A. The skin of Sample B and Sample C had very similar colors, and there was no visible difference or feel in the texture of the fish skin.

[0349] No samples of the liquid solution were collected for color quantification; the prototype was created solely for testing UV radiation and how it affects fish skin.

[0350] Another method for measuring the amount of color in the skin can then be used by breaking down a portion of the fish skin in an enzyme and measuring the amount of color in the solution.

[0351] As a means of comparing the color fastness of the crosslinked samples, color bleeding was performed on a piece of each sample by placing the sample in a base and acid (acid / base) solution at 37°C. Additionally, the samples were compared to other prototypes that had previously been colored by making different mordants and 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] 20A-20D show a comparison of the improvement in color retention due to chemical crosslinking by UV irradiation. FIG. 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. FIG. 20B shows fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after immersion in an acid / base solution of the same concentration for 24 hours. FIG. 20C shows fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after immersion in an acid / base solution of the same concentration for 48 hours. Also shown in Figure 20CD are fragments 20-C, 20-B, and 20-A in dishes 2030, 2020, and 2010, respectively, after 72 hours of immersion in acid / base solutions of the same concentration. As can be seen, the color fastness of fragments 20-B and 20-A exposed to UV radiation was significantly improved over fragment 20-C, which was similarly colored but not exposed to UV radiation. This is especially true after 48 and 72 hours, respectively, in the acid / base solution, as can be seen in Figures 20C and 20D. After both 48 and 72 hours, fragments 20-B and 20-C still maintained some color, but after 72 hours, the uncrosslinked fragment 20-C had turned almost white or reverted to its original color. Comparison of fragments 20-B and 20-A at both 48 and 72 hours indicates that meta-staining of sample B, in which the fish skin was stained with UV radiation while in the MB-based color solution, appears to result in fish skin that is slightly more colorfast when exposed to acid / base solutions. That is, the color of fragment 20-B appeared slightly darker than fragment 20-A after 48 and 72 hours of immersion in the acid / base solution.

[0353] Other embodiments and exemplary methods may include, but are not limited to, changing the colorant, the intensity of the UV radiation, changing the wavelength or wavelength range of the UV radiation, changing the staining time, changing the staining density, and changing the time of exposure to UV radiation.

[0354] Based on these examples and the described embodiments, it has been discovered and shown by the inventors that the properties of a pigmented skin substitute, such as a pigmented scaffold material, can be improved, including increasing the fastness of the colorant that colors the skin substitute, increasing the mechanical material properties of the skin substitute, increasing the resistance of the skin substitute to enzymatic and chemical degradation, and extending the life of the colorant applied to the skin substitute under biological conditions, such as a treated wound.

[0355] Possible combinations of embodiments and features The present disclosure provides various examples, embodiments, and features, which should be understood as being combinable with other examples, embodiments, or features described herein, unless expressly stated otherwise or mutually exclusive.

[0356] In addition to the above, further embodiments and examples include:

[0357] 1. A tissue regeneration wound treatment comprising a skin substitute and a colorant applied to the skin substitute, the colorant being a biocompatible colorant that decomposes upon attack by proteases within the treated wound.

[0358] 2. Tissue regeneration wound treatment according to any one or combination of 1 above or 3 to 14 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.

[0359] 3. Tissue regeneration wound treatment according to any one or combination of 1 to 2 above or 4 to 14 below, wherein the skin substitute is an autologous skin graft, a syngeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft.

[0360] 4. Tissue regeneration wound treatment according to any one or combination of 1 to 3 above or 5 to 14 below, wherein the skin substitute comprises a scaffold material.

[0361] 5. A tissue regeneration wound treatment according to any one or combination of 1 to 4 above or 6 to 14 below, wherein the skin substitute comprises a scaffold material comprising an extracellular matrix product.

[0362] 6. A tissue regeneration wound treatment according to any one or combination of 1-5 above or 7-14 below, wherein the extracellular matrix product is in the form of particles, sheets, or meshes.

[0363] 7. A tissue regeneration wound treatment according to any one or combination of 1 to 6 above or 8 to 14 below, wherein the skin substitute is a scaffold material comprising intact decellularized fish skin, and the intact decellularized fish skin comprises an extracellular matrix material.

[0364] 8. A tissue regeneration wound treatment according to any one or combination of 1-7 above or 9-14 below, wherein the wound treatment is crosslinked before, after, or during the addition of a coloring agent to the skin substitute.

[0365] 9. A tissue regeneration wound treatment according to any one or combination of 1 to 8 above or 10 to 14 below, wherein the coloring agent comprises a thiazine dye, a triarylmethane dye, or a combination of a thiazine dye and a triarylmethane dye.

[0366] 10. A tissue regeneration wound treatment according to any one or combination of 1 to 9 above or 11 to 14 below, wherein the staining agent comprises methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV).

[0367] 11. A tissue regeneration wound treatment according to any one or combination of 1 to 10 above or 12 to 14 below, wherein the skin substitute is freeze-dried and a colorant is added to the skin substitute prior to freeze-drying or re-freeze-drying the skin substitute.

[0368] 12. A tissue regeneration wound treatment according to any one or combination of 1 to 11 above or 13 to 14 below, wherein the colorant is added to the skin substitute by dyeing the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the colorant in deionized water or phosphate buffered saline.

[0369] 13. A tissue regenerating wound treatment according to any one or combination of 1 to 12 above or 14 below, characterized in that the coloring agent has one or more of the following properties: antibiotic, antiseptic, antibacterial, antiviral, antifungal, antiparasitic, anti-inflammatory, or antioxidant.

[0370] 14. A tissue regeneration wound treatment according to any one or combination of 1-13 above, wherein the coloring agent does not cause permanent coloring of the wound upon healing.

[0371] 15. A method of wound treatment comprising the steps of providing a tissue regeneration wound treatment according to any one or a combination of 1 to 14 above, applying the tissue regeneration wound treatment to the wound bed, and determining whether the skin substitute has been degraded within the wound by protease attack by determining a change in color of the colorant.

[0372] 16. A method of producing a tissue regeneration wound treatment, the method comprising the steps of providing a skin substitute and adding a colorant to the skin substitute, the colorant being a biocompatible colorant that degrades upon attack by proteases within the treated wound.

[0373] 17. The method according to any one or combination of 16 or 18 to 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, a syngeneic 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 or combination of 16 to 17 above or 19 to 20 below, 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. The method according to any one or combination of 16 to 18 or 20 below, wherein the coloring agent comprises methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV).

[0376] 20. The method according to any one or combination of 16 to 19 above, wherein the colorant is added to the skin substitute by dyeing the skin substitute with a dye solution containing 0.01% to 0.0001% by weight of the colorant in deionized water or phosphate buffered saline.

[0377] A brief list of defined terms To aid in understanding the scope and content of the foregoing written description and appended claims, several selected terms are directly defined below.

[0378] As used herein, the term "base material" may include any material known in the art that may act as a vehicle for a therapeutic agent and, additionally or alternatively, may allow and / or passively regulate moisture at and / or around the wound.

[0379] The term "biocompatible polymer" refers to a polymeric material that is not harmful to the human body. Biocompatible polymers include synthetic or natural polymeric 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 when in direct contact with a wound site.

[0380] As used herein, the degree of "Echelon" refers to the location and / or type of medical care provided to a military member. Echelon I refers to treatment by combat medics as well as self-help and peer-assisted treatment administered away from the battlefield or Echelon II personnel offices / facilities. Echelon II refers to advanced trauma care by physicians, physician assistants, or other qualified medical personnel, Echelon II care is often administered in field hospitals. Echelon III refers to care provided at the corps level and typically includes reconstructive and definitive surgery to save life, limb, and sight, which may be provided in field hospitals equipped with the necessary facilities. Echelon IV refers to complex surgery and extended convalescence (e.g., greater than two weeks), generally provided in a local permanent hospital. Echelon V refers to injuries and / or procedures requiring extensive rehabilitation and convalescent care, Echelon V care is administered in permanent hospitals in the continental United States. The Echelon System described above relates particularly to military personnel and treatment scenarios, but may be analogized to any type of treatment location and / or treatment scenario in civilian and / or local law enforcement scenarios, as appropriate.

[0381] The term "wound" as used herein is intended to encompass tissue damage generally. Thus, the term "wound" includes injuries that cause cuts, tears, and / or destruction of the skin, such as, for example, lacerations, abrasions, incisions, punctures, stripping, or other similar injuries. Wounds may be described by either the size, shape, or scale of the wound. For example, a paper cut is an example of a small straight incision with a relatively small scale, whereas a concussive blast resulting in a large laceration covering one or more body parts is an example of a larger wound with a larger scale. However, each of the aforementioned examples is included 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. Thus, the term "wound" is intended to include a combination of multiple different wounds. For example, a traumatic cut from an explosion may generally be referred to as a wound, even though it is a collection of various lacerations, abrasions, avulsions, and punctures. Furthermore, any underlying tissue damage resulting from an explosion from the aforementioned explosion may further be included within the understanding of this reference to a wound. The term "wound" is also intended to include tissue injuries caused by burn injuries (e.g., thermal and / or chemical burns). Furthermore, the term "wound" is also intended to include injuries resulting from, for example, diabetic foot ulcers, venous leg ulcers, surgery, bedsores, and other causes.

[0383] A "traumatic wound" as used herein refers to any wound resulting from a physical injury that damages both the skin and the underlying tissue. A gunshot wound is one non-limiting example of a traumatic wound because it causes the skin to puncture (i.e., break) and rupture or otherwise damage the underlying tissue. As another non-limiting example, a concussion or explosion generally results in a traumatic wound(s). Many, but not all, wounds sustained during war may be described as traumatic wounds due to the nature of war and war-related injuries. A "traumatic wound" may include bleeding wounds, wounds that expose 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, burn wounds, and soft tissue repair. Unlike other animal-derived products, fish skin requires gentle processing that preserves structure and bioactive components without the risk of disease transmission to humans. Omega3 Wound has demonstrated advantages over porcine small intestine-derived scaffolds in terms of faster wound closure and rapid healing times. Fish skin grafts have been used in numerous chronic and acute wounds of various etiologies and have shown robust safety and efficacy. Given the complex and hostile environment of war, a holistic approach combining advanced wound care techniques with infection prevention measures should be taken. It is important that new technologies consider and address the needs of soldiers and medical personnel.

[0385] Various changes and / or modifications of the features of the invention exemplified herein, and additional applications of the principles exemplified herein, which would occur to one skilled in the art and possessing this disclosure, may be made to the exemplified 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. Thus, although various aspects and embodiments are disclosed herein, other aspects and embodiments are contemplated. Although many methods and components similar or equivalent to those described herein can be used to implement the embodiments of the present disclosure, only specific components and methods are described herein.

[0386] It is also understood that systems, devices, products, kits, methods, and / or processes according to certain embodiments of the present disclosure may include, incorporate, or otherwise comprise properties, features (e.g., components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Thus, various features of a particular embodiment may be compatible with, combined with, included in, and / or incorporated in other embodiments of the present disclosure. Thus, the disclosure of a particular feature with respect to a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of said feature to the particular embodiment. Rather, it is understood that other embodiments may also include said features, members, elements, parts, and / or portions without necessarily departing from the scope of the present disclosure.

[0387] Furthermore, unless a feature is described as required in combination with another feature, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. Moreover, various well-known aspects of example systems, methods, devices, and the like are not described in particular detail herein to avoid obscuring aspects of the example embodiments. However, such aspects are contemplated herein.

[0388] It should be understood that not necessarily all objects or advantages are achieved under the embodiments of the present disclosure. Those skilled in the art will recognize that the exoskeletons and methods for making the exoskeletons may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without achieving other objects or advantages as taught or suggested herein.

[0389] Those skilled in the art will recognize some interchangeability of the various disclosed features, and in addition to the variations described herein, one of skill in the art will be able to mix and match other known equivalents for each feature to construct an exoskeleton and utilize methods of making an exoskeleton based on the principles of the present disclosure.

[0390] While the present disclosure describes certain exemplary embodiments and examples of a passive lumbar exoskeleton, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed passive lumbar exoskeleton embodiments to other alternative embodiments and / or uses of the present disclosure and obvious modifications and equivalents thereof. It is intended that the present disclosure should not be limited by the disclosed embodiments described above, but may be extended to other applications that may utilize the features described herein.

Claims

1. Skin substitutes; and a colorant added to the skin substitute, the colorant being biocompatible and degrading upon protease attack, enzymatic degradation, chemical degradation, or hydrolysis within the treated wound; 1. A tissue regeneration wound treatment composition comprising: A tissue regenerating wound treatment composition, wherein the skin substitute comprises a scaffold material, the scaffold material comprising a decellularized extracellular matrix material suitable for infiltration and ingrowth by host cells.

2. 10. The tissue regeneration wound treatment composition of claim 1, wherein the skin substitute comprises a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological skin substitute and a synthetic skin substitute.

3. 3. The tissue regeneration wound treatment composition of claim 1 or 2, wherein the skin substitute comprises an autologous skin graft, a syngeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft.

4. A tissue regeneration wound treatment composition described in 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 ingrowth by the host cells, endothelial cells and / or epithelial cells and / or fibroblasts.

5. The tissue regenerating wound treatment composition of any one of claims 1 to 4, wherein the scaffold material comprises an extracellular matrix product.

6. The tissue regenerating wound treatment composition of any one of claims 1 to 5, wherein the extracellular matrix product is in the form of particles, or a sheet, or a mesh.

7. the scaffold material comprises intact decellularized fish skin; The tissue regeneration wound treatment composition of any one of claims 1 to 6, wherein the intact decellularized fish skin comprises an extracellular matrix material.

8. A tissue regeneration wound treatment composition described in any one of claims 1 to 7, wherein the tissue regeneration wound treatment composition is crosslinked.

9. 9. The tissue regeneration wound treatment composition of claim 1, wherein the colorant comprises a thiazine dye, a triarylmethane dye, or a combination of a thiazine dye and a triarylmethane dye.

10. 10. The tissue regeneration wound treatment composition of any one of claims 1 to 9, wherein the coloring agent comprises methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV).

11. The tissue regeneration wound treatment composition of any one of claims 1 to 10, wherein the skin substitute and the colorant added to the skin substitute are in lyophilized form.

12. 12. The tissue regeneration wound treatment composition of any one of claims 1 to 11, wherein the colorant has one or more of the following properties: antibiotic, antiseptic, antibacterial, antiviral, antifungal, antiparasitic, anti-inflammatory, or antioxidant.

13. A tissue regeneration wound treatment composition according to any one of claims 1 to 12, wherein the colouring agent does not cause permanent colouring of the wound as it heals.

14. 1. A method for determining degradation of a skin substitute, comprising: detecting a change in color of the colorant of the tissue regenerating wound treatment composition of claims 1 to 13 applied to the wound bed; determining that the skin substitute is degraded in the wound by protease attack, enzymatic degradation, chemical degradation, or hydrolysis when said color change is detected; A method comprising:

15. 1. A method of making a tissue regenerating wound treatment composition, said method comprising: Providing a skin substitute; adding a colorant to the skin substitute, the colorant being a biocompatible colorant that degrades upon protease attack, enzymatic degradation, chemical degradation, or hydrolysis within the treated wound; wherein the skin substitute comprises a scaffold material, the scaffold material comprising a decellularized extracellular matrix material suitable for infiltration and ingrowth by host cells.

16. the skin substitute is a biological skin substitute, a synthetic skin substitute, or a hybrid of a biological and a synthetic skin substitute; and / or the skin substitute is an autologous skin graft, a syngeneic skin graft, an allogeneic skin graft, a xenogeneic skin graft, or a synthetic skin graft; and / or The method of claim 15 , wherein the scaffolding material comprises an extracellular matrix product.

17. the scaffold material comprises intact decellularized fish skin; 17. The method of claim 15 or 16, wherein the intact decellularized fish skin comprises extracellular matrix material.

18. 18. The method of any one of claims 15 to 17, wherein the colorant comprises methylene blue (MB), gentian violet (GV), or a combination of methylene blue (MB) and gentian violet (GV).

19. 19. The method of any one of claims 15 to 18, wherein the coloring agent is applied to the skin substitute by dyeing 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.

20. A method described in any one of claims 15 to 19, wherein the tissue regeneration wound treatment composition is crosslinked before, after, or simultaneously with the addition of the colorant to the skin substitute.

21. A method described in any one of claims 15 to 20, wherein the skin substitute is freeze-dried and the colorant is added to the skin substitute before freeze-drying or re-freeze-drying the skin substitute.

22. A tissue regeneration wound treatment composition described in any one of claims 1 to 13, wherein the colorant is a biocompatible colorant that decomposes when attacked by a protease.

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