Wound treatments and methods for stabilizing, protecting, and treating wounds - Patents.com

Abstract

Wound treatments and methods of treating wounds are provided that include particles of decellularized fish skin, where a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound.

Description

[Technical field]

[0001] The present disclosure relates generally to wound treatments for stabilizing, protecting, and / or healing damaged tissue. [Background technology]

[0002] Trauma and resulting wounds are an unsolved problem for medical personnel and first responders in military and emergency situations, particularly in situations where there is risk of significant blood loss, loss of limb, infection, and / or other trauma. Blast injuries from explosions are a contributing factor in up to three-quarters of all field-related military injuries, which often cause complex soft tissue loss and result in infection. For example, wounds resulting from explosives or gunfire often result in significant blood loss, and therefore tourniquets are traditionally applied to control blood flow (e.g., reduce or completely stop blood flow), which carries a high risk of tissue necrosis and amputation. However, tourniquets, when used alone, can cause downstream tissue death and result in amputation. In these situations, it is clear that improvements beyond the tourniquet are needed.

[0003] Some wound treatments, such as in battlefield and first responder emergencies, include hemostatic agents, such as clotting powders, hydrated aluminum silicates (e.g., kaolin), and chemical cauterizing agents (e.g., silver nitrate and trichloroacetic acid). Such hemostatic agents act in a variety of ways to slow or stop bleeding at the wound site. For example, some clotting powders incorporate granular chitosan to slow and / or stop bleeding. Chitosan is derived from the chitin-rich shells of crustaceans, and its hemostatic action is known to be the result of direct electrostatic interactions between the negatively charged red blood cell membrane and the positively charged chitosan, independent of the classical clotting pathway.

[0004] On the other hand, some hemostatic agents rely on the classical clotting pathway and act to overactivate blood clotting factors in the blood to shorten clotting time. Hydrated aluminum silicates such as kaolin are known to act in this way. Chemical cautery agents cause wound closure in a destructive manner, via chemical reactions that fuse tissues.

[0005] Additional compounds can be administered on-site to stabilize and / or protect the traumatic wound and to help treat and / or prevent potential complications arising therefrom, including anti-inflammatory compounds, pain relievers, and antibiotic ointments. However, currently available treatment options are focused on stabilizing the patient and do not act to preserve tissue integrity.

[0006] Failure to preserve the tissue integrity of traumatic wounds often leads to tissue desiccation and deterioration. Furthermore, many of the current treatments fail to provide an adequate barrier against subsequent infection and / or keep the wound away from dirt and harmful pathogens. Furthermore, current treatments are ill-equipped to accommodate long-term in-situ care. In short, improved approaches to stabilizing and / or protecting wounds, especially in the field, are needed to preserve the patient and the wound for further subsequent care.

[0007] In other settings, including long-term wound care environments such as operating rooms, trauma, severe burns, and diabetic patients, existing approaches to wound care are often unsatisfactory at best. For example, negative pressure wound therapy ("NPWT") is often implemented after debridement and is used to promote blood flow to the wound, control edema, and reduce the presence of proteases, thus leading to improved granulation and revascularization of the wound bed.

[0008] However, NPWT often suffers from disadvantages in terms of technical issues, inability to precisely control the pressure applied in geometrically challenging wounds or wounds near or in anatomically sensitive areas where an adhesive seal is difficult to obtain, bleeding (which can be difficult to assess due to interference from the dressing), skin irritation, infection, discomfort, and ingrowth of granulation tissue into the dressing material. Additionally, NPWT machines are typically bulky and dependent on electricity.

[0009] Another commonly prescribed treatment for wounds is hyperbaric oxygen therapy, which consists of exposing the patient to high pressure (2.0-2.5 atm) while breathing pure oxygen, with the goal of delivering oxygen to the wound bed in a way that promotes wound healing. It is thought to work by increasing the partial pressure of oxygen, forcing more oxygen into the bloodstream than would be possible under normal conditions. While certain clinicians value this approach, hyperbaric oxygen therapy is inherently expensive (requiring dedicated hyperbaric equipment and trained staff), time-consuming (requiring daily 60-90 minute sessions for many days), and has known risks, including the risk of seizures.

[0010] Other traditional approaches to wound therapy include the "ladder approach," which begins with stabilizing the wound bed and ultimately includes grafts and tissue transfers to provide functional and aesthetic outcomes. However, certain wounds, such as traumatic blast wounds, are not amenable to the traditional ladder approach, as tissue damage often extends beyond the visible wound.

[0011] The traditional reconstructive ladder approach has been adapted by trauma surgeons into a "reconstructive elevator," facilitating rapid advancement with available technology and prioritizing functional and aesthetic outcomes. This, in some cases, results in patients skipping simpler options in order to obtain optimal outcomes. More recently, however, treatment options have benefited from the introduction of new technologies, such as dermal regeneration templates ("DRT"), which allow for more efficient treatment of complex wounds using simpler techniques. However, certain existing DRT options are not ideal for prolonged field care ("PFC") due to the potential for hematoma formation and clinical infection due to shear forces generated during patient transport.

[0012] Blast wounds, and other wounds such as large and complex wounds, are often problematic due to the lack of donor tissue for such wounds and for patient stability. Furthermore, in PFC and first response situations where time is critical, grafts may be impractical because they are difficult to obtain, time-consuming, and resource-intensive. Thus, there is a need for new DRT techniques that are not as sensitive to shear forces during PFC and / or first response situations as existing approaches.

[0013] Wound care is also complicated by the complexity of wound geometries, which may be incisions, lacerations, abrasions, punctures, avulsions, cuts, or combinations thereof, with tunnel wounds, encroaching wounds, cavities, etc., and the specific nature of the explosion, traumatic accident, or other injury adds to the complexity.

[0014] Existing approaches to wound therapy and treatment involve placing sheets of material cut to a specific size on a wound bed, however, what is needed is a wound treatment that can be easily configured to the specific geometry of a patient's wound or wounds without cutting the sheet material to a specific size, which may be a combination of different types of wounds having different conditions and needs for different areas of the wound bed, while being configured to promote tissue healing, e.g., regeneration and / or regrowth, by providing, in embodiments, one or more scaffold materials.

[0015] Wound care can nevertheless be further complicated by the type of wound, e.g., diabetic foot ulcers (DFUs), venous leg ulcers (VFUs), surgical wounds, pressure ulcers (PUs), burns, traumatic wounds, combinations thereof, etc.

[0016] Existing approaches to wound care involve, for example, the use of human placenta-based connective tissue matrix materials, however, these materials have inherent limitations due to the limited availability of placental raw materials and the safety of donating tissue of human origin.

[0017] In view of the above, the inventors have discovered that there is a need for a wound treatment that is rugged and robust, lightweight, compact, easy to transport and handle, has low reliance on external power or specialized equipment, is modular, and is interoperable with current approaches to care. Additionally, there is a need for a wound treatment that withstands shear forces. Additionally, the inventors have discovered that there is a need for a wound treatment that is configured to more easily and more effectively adapt to the different geometries of different wound types. Additionally, there is a need for a wound treatment that is sustainable, scalable, and safe for human use. Summary of the Invention

[0018] A wound treatment agent is provided that includes particles of decellularized fish skin, the particles having a maximum dimension within a range between a predetermined size threshold and a minimum size threshold that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound.

[0019] A method of treating a wound is provided, the method comprising the steps of providing shredded decellularized fish skin particles by shrinking a sheet of decellularized fish skin, applying the shredded decellularized fish skin particles to a wound bed, and covering the wound bed with a dressing.

[0020] A method of treating a wound is provided that includes particles of decellularized fish skin, the particles having a maximum dimension within a range between a predetermined size threshold and a minimum size threshold that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound, the method comprising applying the particles of decellularized fish skin to a wound bed and covering the wound bed with a dressing.

[0021] A method of providing wound treatment is provided, the method comprising the steps of providing one or more sheets of decellularized fish skin and comminuting the one or more sheets of decellularized fish skin into particles.

[0022] A wound treatment is provided comprising particles of decellularized fish skin, wherein a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a range between a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound.

[0023] A method of treating a wound, the method comprising the steps of: providing particles of decellularized fish skin, wherein at least a predetermined percentage of a first portion of the particles of decellularized fish skin have a maximum dimension within a range between a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound; applying the particles of decellularized fish skin to a wound bed; and covering the wound bed with a dressing.

[0024] A method of providing a wound treatment, the method comprising the steps of providing one or more sheets of decellularized fish skin, and chopping or grinding the one or more sheets of decellularized fish skin into particles of decellularized fish skin such that a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound.

[0025] The wound treatment embodiments and treatment methods of the present disclosure advantageously solve one or more of the problems in the art of wound treatment for stabilizing, protecting, and / or healing wounds by providing a DRT that is more easily and / or effectively deployed in situations such as the battlefield, first response situations such as automobile accidents, operating rooms, and other wound sites where wounds are treated. Advantageously, wound treatment embodiments may be advantageously configured to resist shear forces, facilitating transport of a patient between one location, such as the battlefield, first response situation, or assisted living environment, and another location, such as a clinical site, and vice versa.

[0026] Furthermore, embodiments of the present disclosure also extend to dressings for wound treatment. Exemplary dressings can include or be configured to cooperate with shredded, decellularized fish skin particles. The shredded, decellularized fish skin particles can be rehydrated before application to the wound site. The dressing can also include a covering for fixing the shredded, decellularized fish skin particles in particulate form at the wound site, such as a deep wound, which can compress the shredded, decellularized fish skin particles into the deep wound.

[0027] Thus, wound treatments, dressings, kits and methods for stabilizing, protecting and / or healing wounds are disclosed.

[0028] In embodiments, chopped, decellularized fish skin particles may be used in combination with a sheet-based decellularized fish skin scaffold.

[0029] In other embodiments, a temporary wound treatment is provided that includes ground decellularized fish skin in particulate form. Preferably, the particulate form of the ground decellularized fish skin is configured to minimize cellular scaffold formation at the wound site during the temporary wound treatment. This facilitates, in embodiments, a temporary wound treatment that stabilizes and / or protects the wound, e.g., in preparation for receiving a subsequent or higher level of care. For example, the temporary wound treatment may be configured to protect and preserve the wound until the clinician removes the wound treatment for further treatment. By minimizing cellular scaffold formation, the temporary wound treatment may be removed without compromising the wound or removing necessary cell and vascular growth and structure.

[0030] In some embodiments, the temporary wound treatment further includes a temporary dressing configured and arranged to deliver the comminuted decellularized fish skin in particulate form to the wound. The temporary dressing can include, for example, a contact layer configured to contact the wound and hold the comminuted decellularized fish skin at the wound, and an outer cover associated with the contact layer and configured to hold the contact layer at the wound.

[0031] 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.

[0032] 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. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 2 is a plan view of a wound treatment according to an embodiment comprising a first size of chopped, decellularized fish skin particles. [Figure 1B] FIG. 2 is a plan view of a wound treatment according to another embodiment including chopped, decellularized fish skin particles of a second size. [Figure 1C] FIG. 13 is a plan view of a wound treatment according to another embodiment including a third size of chopped, decellularized fish skin particles. [Figure 2A] FIG. 1 is a diagram of a method of treating a wound according to an embodiment of applying dry shredded, decellularized fish skin particles. [Figure 2B] FIG. 1 is a diagram of a method of treating a wound according to an embodiment of applying moistened shredded, decellularized fish skin particles. [Figure 3A] FIG. 1 is a perspective view of a wound bed prepared for treatment with a wound treatment according to an embodiment. [Figure 3B] FIG. 3B is a perspective view of the application of dry shredded, decellularized fish skin particles to the prepared wound bed of FIG. 3A. [Figure 3C] FIG. 3B is a perspective view of the wound bed of FIG. 3A with dried shredded, decellularized fish skin particles applied. [Figure 4A] FIG. 1 is a perspective view of a package containing dry, shredded, decellularized fish skin particles to which liquid has been added, according to an embodiment of a wound treatment. [Figure 4B] FIG. 4B is a perspective view of the package of FIG. 4A with liquid-moistened, chopped, decellularized fish skin particles. [Figure 4C] FIG. 4B is a perspective view of the package of FIG. 4A with chopped, decellularized fish skin particles forming a paste. [Figure 4D] FIG. 4D is a perspective view of a wound prepared for application of the paste of FIG. 4C. [Figure 4E] FIG. 1 is a perspective view of a prepared wound with paste applied with an applicator. [Figure 4F] FIG. 1 is a perspective view of a prepared wound with paste applied. [Figure 5A] FIG. 1 is a perspective view of a wound prepared for application of a wound treatment according to an embodiment. [Figure 5B] FIG. 1 is a perspective view of a prepared wound with a wound treatment applied. [Figure 5C] FIG. 1 is a perspective view of a prepared wound with a wound treatment applied and a wound treatment scaffold sheet applied. [Figure 6A] FIG. 1 is a perspective view of a wound prepared for application of a wound treatment according to an embodiment. [Figure 6B] FIG. 1 is a perspective view of a prepared wound with a wound treatment applied. [Figure 6C] FIG. 2 is a perspective view of a prepared wound following application of a wound treatment. [Figure 7A] FIG. 1 is a perspective view of a wound prepared for application of a wound treatment according to an embodiment. [Figure 7B] FIG. 1 is a perspective view of a prepared wound with a wound treatment applied. [Figure 7C] FIG. 1 is a perspective view of a prepared wound with a wound treatment applied and a wound treatment scaffold sheet applied. [Figure 8] Illustrates a sample of decellularized fish skin scaffold material prior to milling into particulate form. [Figure 9A] 9 illustrates various sized samples of decellularized fish skin scaffold material similar to that shown in FIG. 8. [Figure 9B] FIG. 1 illustrates an exemplary depiction of large particles of pulverized decellularized fish skin resulting from grinding a sheet of decellularized fish skin scaffold material with a hemp grinder, according to an embodiment of the present disclosure. [Figure 9C] FIG. 1 illustrates an exemplary depiction of pulverized decellularized fish skin thread-like, flocculent fibers resulting from grinding a sheet of decellularized fish skin scaffold material with a hemp grinder, according to an embodiment of the present disclosure. [Figure 9D] FIG. 1 illustrates an exemplary depiction of small, powder-like particles of pulverized decellularized fish skin resulting from grinding a sheet of decellularized fish skin scaffold material in a hemp grinder, according to an embodiment of the present disclosure. [Figure 10] FIG. 1 illustrates a schematic cross-sectional view of a temporary dressing for use in retaining crushed decellularized fish skin particles at a wound site to stabilize and / or protect said wound, according to an exemplary treatment of the present disclosure. [Figure 11] FIG. 1 illustrates a schematic cross-sectional view of a sleeve for use with comminuted decellularized fish skin in particulate form to stabilize and / or protect a wound, according to an exemplary treatment of the present disclosure. [Figure 12] FIG. 1 is a diagram of an exemplary kit according to an embodiment of the present disclosure, the components of which can be used to stabilize and / or protect a wound. [Figure 13] FIG. 1 illustrates a diagram of an exemplary method for stabilizing and / or protecting a wound using crushed decellularized fish skin particles, according to an embodiment of the present disclosure.

[0034] 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 be limiting in scope but are intended 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

[0035] 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:

[0036] 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.

[0037] The references used are provided for convenience only and therefore do not define the scope of protection or embodiments.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 longer. In certain embodiments, the fish skin is stored in the buffer at a temperature of about 4° C.

[0045] 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.

[0046] The term "extracellular matrix" or "ECM" as used herein refers to the non-cellular tissue material present within fish skin that provides structural support to skin cells in addition to performing a variety of other important functions. ECM as described herein does not include matrix material that is entirely composed or reconstituted from extracted, purified, or separated ECM components (e.g., collagen).

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 gently pouring off the solution).

[0052] 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.

[0053] 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.

[0054] 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 and decellularization are 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 7.5 and 8.5. After bleaching and / or bleaching and decellularization, the fish skin is optionally washed with a solution containing L-glutamine under the pH conditions described above.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] The resulting product (scaffold material) is a sterile, collagen-based matrix with properties that can promote tissue regeneration, repair, and / or replacement (e.g., repair, regeneration, and / or growth of endogenous tissue). The term "scaffold material" refers to a material comprising 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).

[0062] 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.

[0063] 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).

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 accommodate the wound geometry. 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.

[0070] 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 cell 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.

[0071] In an embodiment, the shredded decellularized fish skin particles have a maximum dimension within a range between a predetermined maximum size threshold and a minimum size threshold 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 sheet of decellularized fish skin as described above, then shredding the sheet of decellularized fish skin, and optionally sieving the shredded particles until the shredded decellularized fish skin particles are within the predetermined minimum and maximum size thresholds.

[0072] The chopped, decellularized fish skin particles may be further configured to resist shear forces given their dimensions, thus allowing for improved wound treatment for patients who move or are moved between locations or sets, or during the patient's normal activities, such as walking during recovery.

[0073] The shredded, decellularized fish skin particles of the embodiments may advantageously be applied locally and / or implanted into a wound to provide a scaffold for cell ingrowth and vascularization, and may further provide additional benefits including tissue scaffolding benefits such as adhesion barrier, soft tissue repair, and dehiscence prevention.

[0074] The embodiments of the present disclosure further include a kit for stabilizing, protecting, and / or healing a wound. An exemplary kit can include a container containing chopped, decellularized fish skin in particulate form for placement on or within a wound and held at the wound site by a contact element. The chopped, decellularized fish skin particles can be configured to be placed on the wound bed in dry or wet form.

[0075] In some embodiments, the container further comprises a contact element configured to contact the wound and hold the shredded decellularized fish skin in particulate form at the wound, and an outer cover configured to hold the contact element and the shredded decellularized fish skin in particulate form at the wound. The container may further comprise a substrate for carrying the shredded decellularized fish skin in particulate form and associating with the contact element. The container may also comprise one or more therapeutic agents, including analgesics, anesthetics, cytokines, growth factors, hemostatic agents, antibiotics, antifungals, hydrating compounds, or combinations thereof.

[0076] The container may further contain a liquid, such as a saline solution of a predetermined concentration, which allows the clinician to moisten the shredded, decellularized fish skin particles prior to or during application to the wound, so as to apply the shredded, decellularized fish skin particles in a wet form, instead of, or in addition to, providing them in a dry form.

[0077]

[0013] Embodiments of the present disclosure further extend to methods for stabilizing, protecting, and / or healing a wound. An exemplary method can include applying shredded decellularized fish skin in particulate form to a wound.

[0078] A method of applying the shredded decellularized fish skin particles may include preparing the wound bed, such as by removing necrotic tissue to obtain a clean wound surface and / or washing to remove debris and exudate; pouring or sprinkling the shredded decellularized fish skin particles onto the wound bed, if the shredded decellularized fish skin particles are applied in dry form; wetting the shredded decellularized fish skin particles with a liquid, such as saline, and then applying the moist shredded decellularized fish skin particles to the wound bed using a finger, tongue depressor, or other surgical instrument, if the shredded decellularized fish skin particles are applied in wet form; and, if necessary, covering and securing the wound bed with a dressing, such as a non-adherent dressing, foam, gauze, wrap, and / or other component.

[0079] In embodiments, application of the shredded, decellularized fish skin particles to a wound provides a scaffolding material that promotes ingrowth of cellular tissue into the shredded, decellularized fish skin particles, hi embodiments, the shredded, decellularized fish skin particles resist shear forces when applied to a wound.

[0080] In embodiments, the clinician may choose to apply the chopped decellularized fish skin particles of the wound treatment embodiment in a dry form, so that the chopped decellularized fish skin particles can be poured onto the wound bed to obtain the desired particle distribution. In other embodiments, the clinician may choose to apply the chopped decellularized fish skin particles in a wet form, so that the resulting paste can be molded to the specific dimensions of the wound bed. In embodiments, a combination of wet and dry application may be used. In other embodiments, a combination of sizes of chopped decellularized fish skin particles may be used.

[0081] As used herein, the term "treatment" is intended to be understood by its common dictionary definition. That is, the term "treatment" broadly includes medical treatment 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 treatment (i.e., in the form of a method or set of prescribed actions) provided, or it can refer to medicine used to preserve or impart certain properties to something.

[0082] 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 drug 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.

[0083] 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).

[0084] 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).

[0085] Other scaffold materials derived from mammalian skin / membrane, such as placenta-based wound treatments, have been subjected to harsh chemical treatments due to concerns over the risk of viral and prion transmission and the risk of allergic or other immune reactions with the use of mammalian scaffold materials in humans. These treatments erase the natural three-dimensional organization of the extracellular components or otherwise cause them to lose their ability to promote wound healing to the same or similar extent as decellularized fish skin.

[0086] On the other hand, 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. Moreover, 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 it maintains the native three-dimensional structure of the extracellular components, although the skin cells are stripped during processing. 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.

[0087] Reconstituted collagen-based materials harvested through harsh physical and chemical treatments also fail to maintain their native three-dimensional structure, especially in the natural context of other native 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 fails to provide an equivalent or similar scaffold for promoting wound healing.

[0088] 1A-1C, wound treatments 100, 110, 120 according to embodiments of the present disclosure are shown and described. The wound treatments 100, 110, 120 include one or more shredded, decellularized fish skin particles 102, 112, 122.

[0089] In an embodiment, the shredded decellularized fish skin particles have a maximum dimension within a range between a predetermined maximum size threshold and a minimum size threshold 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, may be smaller than a maximum size, such as 1 mm, and larger than a minimum size, such as the size at which the ECM is destroyed.

[0090] In embodiments, the shredded decellularized fish skin particles are obtained by providing a sheet of decellularized fish skin as described above, then shredding or crushing the sheet of decellularized fish skin, and, in embodiments, sieving the shredded particles until the shredded decellularized fish skin particles are entirely or substantially within the predetermined minimum and maximum size thresholds. In embodiments, the shredded decellularized fish skin particles are substantially within the predetermined minimum and maximum size thresholds when approximately 75% or more of the particles have a maximum dimension that falls between the predetermined minimum and maximum size thresholds, in embodiments when approximately 90% or more of the particles have a maximum dimension that falls between the predetermined minimum and maximum size thresholds, in embodiments when approximately 95% or more of the particles have a maximum dimension that falls between the predetermined minimum and maximum size thresholds, or other percentages as appropriate.

[0091] The chopped decellularized fishskin particles 102 are advantageously less than 1 mm in size. That is, the maximum of one or more of the thickness, length, width, or other measurements of the particulated decellularized fishskin particles 102, i.e., thickness, length, and width, is less than 1 mm. In an embodiment, the particulated decellularized fishskin particles 122 are less than 1 mm on average, or according to any suitable predetermined threshold. In other embodiments, all of the particulated decellularized fishskin particles 122 are less than 1 mm.

[0092] In an embodiment, the particulate decellularized fish skin particles 102 are no larger than 1 mm at any point, which advantageously allows for maximum moldability of the particulate decellularized fish skin particles to fit snugly within the wound.

[0093] In other embodiments, the size of the particulate decellularized fishskin particles 112 is 1-2 mm. That is, the maximum value of one or more of the thickness, length, width, or other measurements of the particulate decellularized fishskin particles 102, i.e., thickness, length, and width, is less than 2 mm and more than 1 mm. Due to their size, the particulate decellularized fishskin particles 112 can advantageously retain the ECM structure and still be easily handled by practitioners such as clinicians.

[0094] In another embodiment, the size of the particulate decellularized fishskin particles 122 is greater than 2 mm. That is, one or more of the thickness, length, width, or other measurements of the particulate decellularized fishskin particles 122 is greater than 2 mm, that is, the maximum of the thickness, length, and width is greater than 2 mm. In an embodiment, the particulate decellularized fishskin particles 122 are greater than 2 mm on average or according to any suitable predetermined threshold. In another embodiment, all of the particulate decellularized fishskin particles 122 are greater than 2 mm. In another embodiment, the predetermined size threshold is not related to the thickness of the particles of the decellularized fishskin.

[0095] The particulate decellularized fish skin particles 102, 112, 122 may, in embodiments, be obtained by grinding sheet-based decellularized fish skin material to a desired size using a suitable grinding device, for example a Universal Cutting Mill Pulverisette 19 with a variable speed of 50-700 rpm, available from Fritsch GmbH of Idar-Oberstein, Germany.

[0096] The grinding device or fish skin processing may include the use of one or more sieves or filters configured to separate the ground sheet-based decellularized fish skin material into an appropriate size distribution. For example, a sieve may capture shredded particles larger than a predetermined maximum size threshold while allowing smaller shredded particles to pass through to reach a container. A second sieve may be used to capture shredded particles larger than a predetermined minimum size threshold while allowing smaller shredded particles to pass through to reach a second container, such as a waste container.

[0097] In certain embodiments, the shredded decellularized fishskin particles may have a size distribution of between 2 mm and 2.8 mm (i.e., the maximum of one or more of the thickness, length, width, or other measurements of the shredded decellularized fishskin particles 122 is between 2 mm and 2.8 mm), between 1.4 mm and 1.99 mm (i.e., the maximum of one or more of the thickness, length, width, or other measurements of the shredded decellularized fishskin particles 122 is between 1.4 mm and 1.99 mm), between 1 mm and 1.39 mm (i.e., the maximum of one or more of the thickness, length, width, or other measurements of the shredded decellularized fishskin particles 122 is between 1 mm and 1.39 mm), greater than 1 mm (i.e., the maximum of one or more of the thickness, length, width, or other measurements of the shredded decellularized fishskin particles 122 is greater than 1 mm), or any other suitable dimensions.

[0098] In embodiments of wound treatment and related methods, combinations of two or more different sizes of chopped decellularized fish skin particles are provided in suitable ratios of sizes or thresholds described herein, such as a first size and a second size of 50:50 by volume or weight, a first size and a second size of 60:40 by volume or weight, a first size and a second size of 70:30 by volume or weight, a first size and a second size of 25:75 by volume or weight, a first size and a second size of 80:20 by volume or weight, a first size and a second size of 90:10 by volume or weight, a first size and a second size of 95:5 by volume or weight, a first size and a second size of 97:3 by volume or weight, a first size and a second size of 98:2 by volume or weight, a first size and a second size of 99:1 by volume or weight, or any other suitable ratio.

[0099] Further, in embodiments of the wound treatment and related methods, combinations of three or more different sizes of chopped decellularized fish skin particles may be used, such as a first size, a second size, and a third size in a ratio of 50:25:25 by volume or weight, a first size, a second size, and a third size in a ratio of 60:20:20 by volume or weight, or any other combination. The first, second, and third sizes may be provided in any suitable ratio of sizes or thresholds described herein, such as a first size, second size, and third size of 70:15:15 by volume or weight; a first size, second size, and third size of 80:10:10 by volume or weight; a first size, second size, and third size of 90:5:5 by volume or weight; a first size, second size, and third size of 60:30:10 by volume or weight; a first size, second size, and third size of 70:20:10 by volume or weight; a first size, second size, and third size of 90:9:1 by volume or weight, or any other suitable combination.

[0100] In one or more embodiments, the antiviral and antibacterial properties of the shredded decellularized fish skin particles act to prevent bacterial and / or viral infection at the wound site, thereby reducing potential complications (e.g., wound infection) and / or increasing the variety of applications and / or environments in which embodiments of wound treatments and methods according to the present disclosure may be used.

[0101] Additionally, the anti-inflammatory (or inflammation-modulating) properties of the omega-3 PUFAs in the shredded decellularized fish skin particles may help regulate inflammation at the wound site and, in some embodiments, help stabilize, protect, and / or heal tissue.

[0102] That is, omega-3 PUFAs are found in fish skin and are retained after decellularization processing. Previous studies have shown that omega-3 PUFAs have antiviral and antibacterial properties and also act as regulators of inflammation. The shredded decellularized fish skin particles inherit and retain these healthy properties after processing as described herein, further contributing to the healing properties of the shredded decellularized fish skin particles of the wound treatment and related method embodiments.

[0103] In some embodiments, the kit (or components thereof) can be used for stabilizing, covering, and / or initiating the wound healing process in tunneling / eroding wounds or other traumatic wounds, including, for example, topical management of bleeding wounds (cuts, lacerations, and abrasions) and / or temporary management of severe bleeding or bleeding wounds.

[0104] In some embodiments, further advantages can be realized through the utilization of systems, kits, and / or methods that incorporate two types of decellularized fish skin products (e.g., sheet-based and shredded decellularized fish skin) together for processing for complex soft tissue wounds. These two types of fish skin can be used in combination with each other to serve different application purposes.

[0105] For example, deep, asymmetrical, and eroded wounds can be filled with shredded decellularized fish skin before being secured with a sheet for optimal wound healing, bleeding control, and infection protection during transfer to a higher-level facility. The secondary cover with the sheet-based decellularized fish skin sheet protects the shredded decellularized fish skin particle-based wound treatment from shear forces, etc. during dressing changes and provides an additional bacterial and hemostatic barrier during transfer. As a result, the injured party can begin healing while awaiting transfer to a medical facility, resulting in a higher quality wound bed for subsequent grafting.

[0106] Additionally, kits with shredded decellularized fish skin particles may provide one or more surgical benefits including, for example, providing an early treatment approach to wounds that controls bleeding, stabilizes the wound bed, initiates the skin regeneration process, and provides microbial control; simplifying treatment options for tunneled or encroached wounds that are not physically optimized for addressing with conventional materials; filling deep sacral wounds and pressure ulcers, allowing for the application of smaller skin flaps and increasing the chances of flap success; and temporarily immobilizing wounds in preparation for autografts and / or skin flap creation.

[0107] With reference to FIG. 2A, a method for wound treatment according to an embodiment of the present disclosure is shown and described. Method 200 generally corresponds to dry application of a wound treatment and may include one or more of the following steps, not necessarily in the order shown. Method 200 may include step 202 of preparing a wound bed. The wound bed may be prepared, in embodiments, by cleansing the wound bed, such as removing necrotic tissue and / or washing the wound bed to remove debris and exudate. In embodiments, step 202 of preparing a wound bed includes removing the previously applied wound treatment, including the shredded decellularized fish skin particles, when it is determined that the shredded decellularized fish skin particles are not integrated after a predetermined threshold period. The predetermined threshold period may be 7 days, 10 days, 2 weeks, or any other suitable threshold period.

[0108] Method 200 may further include applying 204 the dried shredded decellularized fish skin particles from a container to the wound bed. Applying 204 the dried shredded decellularized fish skin particles may include any suitable application method, such as pouring, sprinkling, packing, pressing, molding, combinations thereof, etc.

[0109] For example, when performing step 204, the clinician may pour a first layer of the dried shredded decellularized fish skin particles directly from the package onto the wound bed, and then use an applicator, such as a gloved finger, to apply a pinch of the dried shredded decellularized fish skin particles to the complex shape of the wound bed, such as a tunneled or eroded wound. The clinician may apply a sufficient amount of the dried shredded decellularized fish skin particles to substantially or entirely fill the void defined by the wound bed, such as a tunneled wound. In an embodiment, step 204 may include pouring the dried shredded decellularized fish skin particles into a clean or sterile container before applying the dried shredded decellularized fish skin particles to the wound bed in any suitable manner.

[0110] The method 200 may further include a step 206 of covering the wound bed. The step 206 of covering the wound bed may include a covering utilizing or including a non-adherent dressing and optionally a bolster to ensure contact between the macerated decellularized fish skin particles and the wound bed. That is, the step 206 of covering the wound bed may include placing a covering, such as a non-adherent dressing, sufficiently flush against the wound bed to press the macerated decellularized fish skin particles against the wound bed. The non-adherent dressing may be any suitable dressing, such as a synthetic non-woven non-adherent dressing, a woven cotton non-adherent dressing, etc. The covering may include any suitable covering.

[0111] Step 206 may alternatively or additionally include securing the covering with foam or gauze to maintain moisture in the wound bed and manage exudate. Method 200 may further include step 208 of wrapping the wound bed and securing the covering.

[0112] Method 200 may further include step 210 of checking for integration of the chopped decellularized fish skin particles after a predetermined threshold period. For example, the predetermined threshold period may be a period of two weeks that allows sufficient time for the particles to integrate, i.e., promote cell ingrowth and angiogenesis. If integration is determined not to have occurred upon performance of step 210, the previously applied chopped decellularized fish skin particles may be removed and method 200 may be repeated as part of step 202 of preparing the wound bed.

[0113] 2B, a method for wound treatment according to an embodiment of the present disclosure is shown and described. Method 250 generally corresponds to a wet application of a wound treatment and may include one or more of the following steps, not necessarily performed in the order shown: Method 250 may include step 252 of preparing a wound bed, as described above with respect to method 200.

[0114] The method 250 may further include a step 254 of wetting the shredded decellularized fish skin particles with a liquid. The liquid may be any suitable liquid, such as a saline solution of a predetermined concentration. In an embodiment, the liquid may be 0.9% saline, as known to those skilled in the art. Step 254 may include adding a predetermined amount of liquid, such as 1 cc, 2 cc, etc.

[0115] Method 250 may include step 256 of applying moistened shredded decellularized fish skin particles into or onto the wound bed using an applicator. Step 256 may include forming the moistened shredded decellularized fish skin particles into a paste using a gloved finger, a tongue depressor, a surgical instrument, or any other suitable applicator before applying the paste to the wound bed. As with method 250, applying the moistened shredded decellularized fish skin particles may include the clinician applying a sufficient amount of particles to substantially or entirely fill a void defined by the wound bed, such as a tunnel wound. In other embodiments, step 256 includes applying a layer of moistened shredded decellularized fish skin particles onto the wound bed in addition to or instead of filling the void.

[0116] Method 250 may further include step 206 of securing the dressing as described above with respect to step 208, step 258 of covering the wound bed to which the moistened shredded decellularized fish skin particles have been applied as described above with respect to 260, and / or step 262 of checking integrity as described above with respect to step 210 of method 200.

[0117] With reference to Figures 3A-3C, the application of wound treatment according to an embodiment of the present disclosure is shown and described. Figure 3A shows a wound bed WB prepared for wound treatment of the present disclosure. The wound bed WB in Figure 3A is a wound bed having one or more deep regions. As described above with respect to Figures 2A and 2B, the wound bed WB can be prepared by first removing necrotic tissue and washing the wound bed WB to remove debris and exudate.

[0118] Thereafter, wound treatment 300 including one or more dry shredded decellularized fish skin particles (first removed from packaging and added to sterile container C) is applied to the wound bed WB as shown in Figure 3B. Wound treatment 300 may be poured, sprinkled, packed, or otherwise applied, such as using an applicator such as a gloved finger, a surgical instrument, etc. Wound treatment 300 may be added in an amount sufficient to substantially fill one or more deep regions of the wound bed WB.

[0119] FIG. 3C shows a wound bed WB with wound treatment 300 applied to substantially fill one or more deep regions of the wound bed WB. Wound treatment 300 advantageously allows clinicians to fill a wound bed WB with a complex geometry with scaffolding material without the extensive and time-consuming task of cutting sheet-based decellularized fish skin material to size. Furthermore, wound treatment 300 advantageously fills a complex geometry, including one or more deep regions, using a simple application procedure. By providing wound treatment 300, the chopped decellularized fish skin particles advantageously facilitate cell ingrowth throughout the complex geometry of the wound while also providing the antiviral and antibacterial benefits of the omega-3 PUFAs provided thereby.

[0120] 4A-4F, application of a wound treatment according to an embodiment of the present disclosure is shown and described. The wound treatment 400 is applied to the wound bed in a wet or moistened form as described above with respect to the method 250 of FIG. 2B. The wound treatment 400, including the shredded decellularized fish skin particles of an appropriate size distribution as described above, may be provided in a package 402. The package 402 may be formed of any suitable material, such as a polymeric material, suitable for receiving a liquid 450, such as 0.9% saline, within an interior pocket defined by the package 402. The liquid 450 may be delivered using any suitable modality, such as a syringe 452 or other device. In an embodiment, the syringe 452 may define indicia that allow a clinician to provide a predetermined amount of the liquid 450, such as 1 cc or 2 cc. The package 402 may be formed of a transparent or see-through material such that the clinician may view the dried shredded decellularized fish skin particles 400 as the liquid 450 is added. With reference to FIG. 4B, the package 402 may be configured to be opened, such as by peeling back a layer, to allow a clinician access to the moistened fish skin particles 400 therein.

[0121] The moistened shredded decellularized fish skin particles 400 can be formed into a paste 404 using any suitable applicator, such as a gloved finger, as shown in Figure 4C. This can be performed in or on the package 402, on an eye-sterile surface, or any other suitable location.

[0122] Referring to FIG. 4D, a wound bed WB for application of the wound treatment 400 is shown. The wound bed WB may define a complex geometry, such as a tunneled and / or eroded wound, and / or may be arranged such that a sheet-based decellularized fish skin is inappropriate and / or difficult to apply. As seen in FIG. 4E, the paste 404 is applied to the wound bed WB using an applicator APP. The applicator APP may be a gloved finger, a tongue depressor, a surgical instrument, or the like. In an embodiment, the application of the paste 404 includes pressing or packing the paste 404 into the wound bed WB to fill or substantially fill the void defined by the wound bed WB. Due to the size of the chopped decellularized fish skin particles, the paste 404 of the wound treatment 400 is configured to closely conform to the geometry of the wound bed WB.

[0123] As seen in FIG. 4F, the wound bed WB may, in embodiments, be filled up to the skin surface with paste 404. The application of moistened shredded decellularized fish skin particles may advantageously aid in the moldability, shape retention, flexibility, and removability of the wound treatment 400. While existing approaches to wound treatment tend to leak out of place during application, paste 404 has surprisingly been found to stay in place and maintain its shape, thereby improving the effectiveness of the wound treatment. This further reduces wound treatment waste.

[0124] Referring to Figures 5A-5C, a wound bed WB is shown. The wound bed WB in Figure 5A is a medium to large size wound with uneven and deep areas. The wound treatment 500 may be applied to the wound bed WB in a dry or wet state and in an amount sufficient to fill or substantially fill a desired portion of the wound bed. In the embodiment of Figures 5A-5C, a further step of applying a sheet-based decellularized fish skin material 550 is performed. The sheet-based decellularized fish skin material may be a scaffold as taught in U.S. Patent No. 8,613,957. The sheet-based decellularized fish skin material 550 advantageously holds the wound treatment 500 in place while itself promoting wound healing, cell ingrowth, and vascularization. One or more fasteners 552 hold the sheet-based decellularized fish skin material 550 in place relative to the wound bed WB.

[0125] 6A-6C, a wound bed WB is shown. The wound bed WB in FIG. 6A is a deep wound that is not suitable for existing sheet-based scaffolding materials due to its depth. A wound treatment 600 according to an embodiment of the present disclosure can be applied to the wound bed WB using an applicator APP, such as a surgical instrument, such that the wound treatment 600 fills or substantially fills the entire wound bed WB. Due to the size of the chopped decellularized fish skin particles of the wound treatment 600, the wound treatment 600 can be easily, quickly, and effectively applied to the wound bed WB, reducing waste, cost, and time while providing a scaffolding material within the wound bed to promote cell ingrowth throughout the complex wound geometry. As seen in FIG. 6C, new tissue NT is ultimately obtained from the wound treatment 600.

[0126] With reference to Figures 7A-7C, a wound bed WB having an irregular shape is shown. The WB includes multiple wound beds of different shapes and depths, complicating the application of existing wound treatments. As seen in Figure 7B, wound treatment 700 is applied to the multiple wound beds WB as described herein, either dry or wet, such that wound treatment 700 substantially conforms to the complex geometry of the wound bed WB. This can be done with minimal effort, time, and waste, thanks to the dimensions of the shredded decellularized fish skin particles, which despite their small size (which allows them to be packed into the wound bed), provide a scaffolding material that promotes cell ingrowth and vascularization, while also providing healthy omega-3 PUFAs to the wound bed WB.

[0127] Similar to the embodiment of Figures 5A-5C, a further step of applying a sheet-based decellularized fish skin material 750 is performed. The sheet-based decellularized fish skin material 750 may be a scaffold as taught in U.S. Patent No. 8,613,957. The sheet-based decellularized fish skin material 750 advantageously holds the wound treatment 700 in place while advantageously promoting wound healing, cell ingrowth, and vascularization itself. One or more fasteners 752 hold the sheet-based decellularized fish skin material 750 in place relative to the wound bed WB.

[0128] Temporary wound treatment to stabilize and / or protect traumatic wounds In one or more other embodiments, a temporary wound treatment is provided that includes comminuted decellularized fish skin in particulate form. Preferably, the particulate form of the comminuted decellularized fish skin is configured to minimize cellular scaffold formation at the wound site during the temporary wound treatment.

[0129] This facilitates, in embodiments, a temporary wound treatment that stabilizes and / or protects the wound, e.g., in preparation for receiving a subsequent or higher level of care. For example, the temporary wound treatment may be configured to protect and preserve the wound until the clinician removes the wound treatment for further treatment. By minimizing cellular scaffold formation, the temporary wound treatment may be removed without compromising the wound or removing necessary cellular and vascular growth and structure.

[0130] In some embodiments, the temporary wound treatment further includes a temporary dressing configured and arranged to deliver the comminuted decellularized fish skin in particulate form to the wound. The temporary dressing can include, for example, a contact layer configured to contact the wound and hold the comminuted decellularized fish skin at the wound, and an outer cover associated with the contact layer and configured to hold the contact layer at the wound.

[0131] The temporary wound treatment for stabilizing and / or protecting the wound can also include a substrate, such as a biocompatible polymer, infused with or otherwise carrying the crushed decellularized fish skin particles. Additionally or alternatively, the temporary wound treatment for stabilizing and / or protecting the wound can include a compression element associated with the outer cover configured to conform the outer cover to the shape of the wound and / or the shape of the partial or entire limb containing the wound. The compression element can include, for example, a sleeve having an inflatable bladder. In some embodiments, the temporary wound treatment can include a substrate disposed on the bottom or peripheral wall of the sleeve and associated with a contact layer.

[0132] The pulverized decellularized fish skin particles in the temporary wound treatment may be less than about 1 cm in diameter, less than about 0.1 cm in diameter, less than about 10 mm in diameter, less than about 1 mm in diameter, less than about 0.1 mm in diameter, less than about 10 μm in diameter, less than about 1 μm in diameter, or combinations thereof. Additionally or alternatively, the pulverized decellularized fish skin in particulate form may be partially processed, such as by treating with enzyme(s) to reduce the stiffness of the pulverized decellularized fish skin particles. In some embodiments, such partial processing may cause at least a portion of the extracellular matrix material in the partially processed, pulverized decellularized fish skin particles to be cleaved by enzyme(s), increasing the ductility and / or elasticity of the partially processed, pulverized decellularized fish skin particles.

[0133] The embodiments of the present disclosure further include a kit for stabilizing and / or protecting a wound. An exemplary kit can include a container containing pulverized decellularized fish skin in particulate form for placement on or within the wound and held at the wound site by a contact element.

[0134] In some embodiments, the container further comprises a contact element configured to contact the wound and hold the pulverized decellularized fish skin in particulate form at the wound, and an outer cover configured to hold the contact element and the pulverized decellularized fish skin in particulate form at the wound. The container may further comprise a substrate for carrying the pulverized decellularized fish skin in particulate form and associating with the contact element. The container may also comprise one or more therapeutic agents, including analgesics, anesthetics, cytokines, growth factors, hemostatic agents, antibiotics, antifungals, hydrating compounds, or combinations thereof.

[0135]

[0013] Embodiments of the present disclosure further extend to methods for stabilizing and / or protecting a wound. An exemplary method can include applying comminuted decellularized fish skin in particulate form to a wound.

[0136] In some embodiments, a method for stabilizing and / or protecting a wound can include covering the wound with a contact element configured to hold comminuted decellularized fish skin particles at the wound and applying an outer cover to the contact element to secure the contact element to the partial or entire limb containing the wound. In some embodiments, applying the comminuted decellularized fish skin in particulate form to the wound does not promote ingrowth of cellular tissue into the decellularized fish skin in particulate form.

[0137] The embodiments of the present disclosure further extend to a dressing for the treatment of uncleaned and / or undebrided wound sites. An exemplary dressing can include pulverized decellularized fish skin in particulate form. The pulverized decellularized fish skin in particulate form can be rehydrated before application to the wound site. The dressing can also include a covering for fixing the pulverized decellularized fish skin particles in particulate form at the wound site, such as a deep wound, which can compress it into the deep wound.

[0138] Accordingly, temporary wound treatments, dressings, kits and methods for stabilizing and / or protecting wounds are disclosed.

[0139] Current prehospital battlefield trauma care, including extremity hemorrhage, focuses solely on controlling bleeding until the injured soldier can receive definitive care. These care consist of applying compression and a tourniquet, and covering the wound with gauze and hemostatic dressings. However, evacuation times for injured soldiers are expected to increase, and in some instances, soldiers are already deployed to remote locations where evacuation may take days, increasing the need for more advanced pre-evacuation care. In addition to controlling hemorrhage, pre-evacuation care is needed to prevent infection, minimize further tissue loss, protect underlying tissues / organs, reduce ischemia and secondary injury, and reduce pain and distress.

[0140] Preservation of tissue status is important to minimize the loss of damaged tissue (including that surrounding the wound) caused by necrosis, debridement, or amputation, and to maximize the chances that the affected area can recover. If the injury can be promptly and properly treated, there is a greater chance that the affected area can recover while minimizing the loss of structure and / or function at or around the wound site. However, the medical facilities and equipment required to properly treat traumatic wounds and maximize positive outcomes from rehabilitation are often available in permanent hospitals or, in some cases, within centralized field hospitals.

[0141] When traumatic wounds are sustained in remote locations, such as on a battlefield, it is difficult to preserve damaged tissue for the extended periods of time required to transport the wounded soldier to an appropriate treatment facility. In some instances, it may take several days to transport the wounded soldier to an appropriate treatment facility. As a result, traumatic wounds sustained in remote locations are more likely to involve high levels of tissue necrosis, require larger areas of tissue debridement prior to treatment, and lead to amputations and poorer rehabilitation outcomes.

[0142] The embodiments of the present disclosure enable and improve the long-term in situ treatment of wounds, particularly traumatic wounds, burns, and / or amputations, allowing for stabilization and / or preservation of damaged tissue. The embodiments of the present disclosure include treatments and associated kits that reduce the loss of damaged tissue and / or increase the likelihood that the wound can be successfully repaired. This is made possible, at least in part, from the incorporation of a particulate form of pulverized decellularized fish skin at the wound site. When applied to the wound site, the particulate form of pulverized decellularized fish skin acts to stabilize and / or preserve the wound site. The particles can be applied in situ to uncleaned and / or undebrided wounds and can act beneficially to reduce infection, reduce pain associated with the wound, and / or reduce the need for repeated wound debridement. The embodiments disclosed herein can be used to prepare wounds for effective autograft or flap closure, increasing the likelihood of autografting.

[0143] As an advantage, the crushed decellularized fish skin particles are configured, in embodiments, to minimize cellular scaffold formation so that the temporary wound treatment can be removed during subsequent treatments without damaging the newly grown cellular structures.

[0144] As used herein, the term "treatment" is intended to be understood by its common dictionary definition. That is, the term "treatment" broadly includes medical and / or pharmaceutical care 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 set of prescribed actions) or it can refer to a drug used to preserve or impart certain properties to something.

[0145] 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 drug 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.

[0146] As mentioned above, the basis for promoting wound healing is the natural three-dimensional structure of the extracellular matrix components, which acts as a scaffold for cell infiltration and growth. However, before the decellularized fish skin is applied to act as a scaffold, the wound site is usually cleared of necrotic tissue and cleaned. Removal of the dead or damaged skin allows direct access to the subepidermal tissue, on which a new skin layer can form. Therefore, using intact decellularized fish skin in conditions or dimensions that are unfavorable for cell ingrowth, or even destroying the three-dimensional structure of the natural extracellular matrix components, is counter to common sense and seems at first glance undesirable.

[0147] However, surprisingly, mechanical comminution of decellularized fish skin and its subsequent use in particulate form on dirty, undebrided wounds as disclosed herein results in several unexpected advantages: Many of the salutary properties of decellularized fish skin in particulate form are independent of the tissue repair properties of decellularized fish skin when used as a scaffolding material, and the particulate form of comminuted decellularized fish skin, while not substantially repairing tissue, can be utilized to advantage to preserve tissue.

[0148] For example, Applicant believes that application of pulverized decellularized fish skin particles at a wound site in accordance with embodiments of the present disclosure, with or without one or more other elements disclosed herein, provides the unexpected effect of stabilizing and / or protecting the wound and surrounding tissue instead of actively promoting wound healing as a physical scaffold for the infiltration of cells involved in wound healing / repair.

[0149] These surprising and unexpected wound preservation and stabilization properties allow the pulverized decellularized fish skin particles of the embodiments to be utilized in new and previously unanticipated scenarios, and in some embodiments may be used to serve different purposes than previously envisioned for decellularized fish skin scaffolds. For example, the pulverized decellularized fish skin in particulate form may be used as part of a temporary dressing, where the decellularized fish skin particles act to stabilize the wound until further treatment is administered.

[0150] In some embodiments, the crushed decellularized fish skin particles are applied directly to unclean (e.g., uncleaned or undebridement) wounds to stabilize and / or protect the wound until the wound can be cleaned, debrided, and treated in a properly equipped and staffed medical facility. The crushed decellularized fish skin in particulate form is not intended to promote wound healing, particularly as a scaffolding material. Instead, the crushed decellularized fish skin particles are provided to the wound site and later removed during subsequent pre-treatment wound cleansing / debridement. As described herein, the crushed decellularized fish skin particles beneficially improve the hemostatic properties of the wound to manage / reduce blood loss, help maintain a proper moist environment at the wound site to prevent drying from further damage to the tissue, provide antibacterial and antiviral defenses, modulate inflammation, and in some instances reduce pain.

[0151] This is in contrast to other applications and embodiments of decellularized fish skin, which are primarily used as a scaffolding material to promote cell ingrowth for long-term wound healing treatments, where it has particular advantages. If a decellularized fish skin scaffolding is applied to a clean and debrided wound, but is removed during the wound healing process (e.g., after several days), the wound will at least partially reopen as the scaffolding material and all wound healing cells inserted / associated with it will be ripped away from the wound site. Such an action, instead of stabilizing and / or protecting the wound site, may cause further trauma to it, exacerbating the problem.

[0152] On the other hand, the grinding of decellularized fish skin into particle form according to the embodiments transforms the decellularized fish skin scaffold, allowing additional treatment benefits and options, including, for example, use as a hemostatic agent for dirty and / or undebrided wounds, temporarily preserving the wound site and allowing the transport of the injured person to a distant medical facility where the wound can then be treated. The ground decellularized fish skin particles can be easily used in the field without the need for special medical training. The ground decellularized fish skin particles can be included to stabilize and / or protect the wound, with or without a substrate or even as part of a temporary dressing. This allows for improved and / or prolonged care treatment in the field, increasing the chances of rehabilitation of the affected area, among other things.

[0153] Furthermore, pulverization allows for a more dynamic and rapid application of the decellularized fish skin to the wound site than the non-pulverized form, which in some embodiments allows some of the healthy properties of the decellularized fish skin, such as hemostatic, barrier function, and / or analgesic properties, to be more pronounced in the treatment.

[0154] As used herein, the term "comminute" refers to the act of reducing a material into smaller fragments or particles. Comminution can be done by any process or force, including, without limitation, mechanical force (e.g., cutting, shredding, tearing, crushing, shearing, grinding, jet milling, etc.), focused heat (e.g., laser cutting), any process or mechanism for reducing a material into fragments or particles, or a combination thereof. For example, a very sharp blade and / or a low RPM mill can be used to grind the decellularized fish sheet into a particulate form.

[0155] Thus, the term "comminuted" may be used herein to refer to the physical properties of the material that has been subjected to comminution and / or the resulting product. Thus, the term "comminuted decellularized fish skin" as used herein may refer to the physical properties of the decellularized fish skin, i.e., the decellularized fish skin is expressed as smaller fragments or particles, or may additionally or alternatively refer to the resulting product, i.e., the decellularized fish skin, being made into fragments or particles. In some embodiments, it should be understood that comminution reduces the physical size of the decellularized fish skin in at least one dimension, but may not affect the ultrastructure of the particles. That is, some embodiments of the comminuted decellularized fish skin are physically reduced in size but retain their three-dimensional extracellular structure.

[0156] The various morphologies of the milled particles can be separated using sieves or hole filters or otherwise selected based on the method of milling.

[0157] In addition to the above, and as briefly discussed in the Background, soldiers deployed to combat zones are at increased risk for injuries from blast wounds. In future conflicts, combat in remote locations, urban environments, and other settings with reduced communications and air superiority may make transporting wounded soldiers increasingly difficult. This change in combat environment requires alternative solutions for treating injuries in the field, as the current standard of care may not be practical.

[0158] Similarly, wounds sustained in rural areas (e.g., resulting from injuries associated with the use of heavy machinery, hunting accidents, all-terrain vehicle crashes or rollovers, etc.) can benefit from alternative solutions for treating injuries incurred in environments where access to a hospital or specialized medical site is difficult or time-consuming.

[0159] In particular, there is a need for wound care solutions that are rugged and robust (e.g., long half-life, stability in extreme temperatures and conditions), lightweight, compact, easy to transport, simple and quick to handle at the injury site, have little or no dependency on external power sources or specialized equipment, and are modular and interoperable (e.g., capable of being integrated with current care approaches).

[0160] Embodiments of the present disclosure may solve one or more of the problems discussed above and exhibit many, if not all, of the desired properties discussed above. For example, a kit including a source of crushed decellularized fish skin particles may be used in the field to preserve and / or stabilize wounds (e.g., combat-related traumatic wounds).

[0161] For example, crushed decellularized fish skin, when packed into asymmetric deep tissue wounds in injured soldiers, can have applications as a hemostatic agent, antibacterial agent, and initiate dermal regeneration. This can be accomplished in the field and / or in field-based medical facilities close to the injury site, instead of state-of-the-art medical facilities where similar benefits are traditionally achieved.

[0162] In some embodiments, sheets of decellularized fish skin can be used in combination with comminuted decellularized fish skin or alone to provide fast acting hemostatic and antimicrobial properties to cover wound sites. When used in combination, sheet-based comminuted decellularized fish skin can provide benefits not realized by using the sheet-based product alone, such as enhanced granulation of the wound bed, making the wound more suitable for early grafting or flap surgery, and / or ultimately resulting in better functional and aesthetic outcomes, as well as long-term benefits such as improved wound healing rates.

[0163] It should be readily appreciated that the embodiments disclosed herein also have direct application for public use (e.g., shock and trauma centers), particularly in rural areas where suboptimal medical care and / or infrastructure may prevent ideal treatment of traumatic wounds, such as deep tissue wounds.

[0164] Exemplary embodiments Embodiments of the present disclosure provide field-use devices, kits, and compositions for deep tissue wound stabilization, coverage, and / or initiation of tissue regeneration for long-term and ongoing care.

[0165] 8 and 9A, illustrated are exemplary embodiments of decellularized fish skin 800, 900 prior to grinding and / or other processing. An exemplary cross-section of decellularized fish skin 800 made as described in U.S. Pat. No. 8,613,957 is illustrated in FIG. 8 with a given size in relation to a user's gloved hand 802.

[0166] It should be understood that the decellularized fish skin may be ground or processed into various sizes. As shown in Fig. 2A, the multiple decellularized fish skin sheets 800, 900 may be of a size and shape (e.g., rectangular) similar to the decellularized fish skin 800 of Fig. 8, or may have a more uniform dimensional (e.g., square) shape, such as the decellularized fish skin sheet 900 illustrated in Fig. 9A.

[0167] The decellularized fish skin scaffold 800, 900 shown in Figures 8 and 9A 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.

[0168] 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.

[0169] 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.

[0170] In one embodiment, the decellularized fish skin is pulverized by mechanically cutting the sheet of decellularized fish skin into pieces and / or particles with a diameter of less than 1 cm. Alternatively or additionally, the decellularized fish skin is pulverized using a mechanical grinder, such as a cannabis grinder.

[0171] The diameter of the fragments and / or particles resulting from the grinding can be less than about 0.1 cm, less than about 10 mm, less than about 1 mm, less than about 0.1 mm, less than about 10 μm, less than about 1 μm, or combinations thereof. That is, in some embodiments, the diameter of the fragments and / or particles can vary and is defined as a range of sizes between any combination of the aforementioned dimensions. In some embodiments, the particles can be size separated.

[0172] In some embodiments, the size of the particles can be uniform or can be within a uniform or variable size range. For example, the crushed decellularized fish skin particles can be size-selected through one or more sieves or screens that retain bulky particles and allow particles smaller than the sieve mesh size to pass through. The particles that pass through the sieve are within a range of sizes with an upper threshold at the sieve mesh size of the first sieve. In some embodiments, additional (or a series of) size-selecting sieves (one or more) can be used to further refine the particle size.

[0173] For example, a sharp blade mill operating at low RPM can be used to pulverize the decellularized fish skin. The mill can be associated with a hole filter, allowing for relatively reliable size selection of the product. In such an embodiment, scale-up can be facilitated by a temperature-controlled low RPM mill.

[0174] Continuing with the previous example, the decellularized fish skin particles that pass through the first sieve, now defined by an upper threshold size, can be filtered on or through a sieve having a smaller mesh size. The particles that are smaller in size (e.g., pass through the second sieve) have an upper threshold particle size that is smaller than the upper threshold size of the particles retained by the second sieve. The particles captured by the second sieve are now additionally defined by a lower threshold particle size.

[0175] Different particle sizes may provide different benefits and may be selected for different applications based on particle size. Additionally or alternatively, a combination of different particle sizes may be selected to obtain the combined benefits provided by different particle sizes. In some embodiments, the particles are 1 cm, 10 mm, 8 mm (2 1 / 2 mesh), 6.73 mm (3 mesh), 5.66 mm (3 1 / 2 mesh), 4.75 mm (4 mesh), 4.00 mm (5 mesh), 3.36 mm (6 mesh), 2.83 mm (7 mesh), 2.38 mm (8 mesh), 2.00 mm (9 mesh), 1.68 mm (10 mesh), 1.41 mm (12 mesh), 1.19 mm (14 mesh), 1.00 mm (16 mesh), 0.841 mm (20 mesh) , 0.707mm (24 mesh), 0.595mm (28 mesh), 0.500mm (32 mesh), 0.420mm (35 mesh), 0.354mm (42 mesh), 0.297mm (48 mesh), 0.250mm (60 mesh), 0.210mm (65 mesh), 0.177mm (80 mesh), 0.149mm (100 mesh), 0.125mm The sieves may have upper and / or lower thresholds defined by passage through and / or retention by one or more sieves having openings of size 0.088 mm (115 mesh), 0.105 mm (150 mesh), 0.088 mm (170 mesh), 0.074 mm (200 mesh), 0.063 mm (250 mesh), 0.053 mm (270 mesh), 0.044 mm (325 mesh), 0.037 mm (400 mesh), 0.025 mm (500 mesh), openings of a size greater than the above sizes (e.g., greater than 1 cm), openings of a size between any of the above (e.g., any size between 0.025 mm and 10 cm), or openings of a size less than the above sizes (e.g., less than 0.025 mm).

[0176] Thus, after grinding, the ground decellularized fish skin can be of the same and / or different shapes, the same and / or different thickness (and / or the same and / or different width and / or length), pellet shape, flakes, powder (or powdered), suspended as a colloid in a mixture / solution, combinations thereof, and / or any other physical state. In one embodiment, the ground decellularized fish skin is equilibrated in a solution before being applied to a wound and / or used as a temporary dressing. Examples of solutions include saline, water, alcohol, antibiotic solutions, hydrating compounds, and / or any aqueous solution with or without one or more therapeutic agents added (or dissolved), and in some embodiments, the solution can be sterilized.

[0177] In one embodiment, the crushed decellularized fish skin particles are part of a temporary dressing that is applied in / with and / or injected into the surface layer and / or contact layer or substrate at the wound. For purposes of this disclosure, it should be understood that, where appropriate, embodiments disclosing the use of crushed fish skin particles may additionally or alternatively incorporate or use decellularized fish skin or modified decellularized fish skin (e.g., enzyme-treated decellularized fish skin with increased ductility / elasticity). Additionally or alternatively, the crushed decellularized fish skin particles may be administered to the wound as a separate or distinct treatment (e.g., by sprinkling on or covering the wound) prior to application of the temporary dressing.

[0178] Figure 9B illustrates an exemplary depiction of large particles of comminuted decellularized fish skin resulting from grinding a sheet of decellularized fish skin scaffold material with a hemp grinder. Figure 9C illustrates an exemplary depiction of thread-like, flocculent fibers of comminuted decellularized fish skin resulting from grinding a sheet of decellularized fish skin scaffold material with a hemp grinder according to an embodiment of the present disclosure. Figure 9D illustrates an exemplary depiction of small, powder-like particles of comminuted decellularized fish skin resulting from grinding a sheet of decellularized fish skin scaffold material with a hemp grinder.

[0179] Referring now to FIG. 10, a temporary dressing 1000 is illustrated. The temporary dressing 1000 is configured and arranged to deliver crushed decellularized fish skin particles 1004 to a wound. The crushed decellularized fish skin particles 1004 may be delivered and / or retained to the wound by the inner surface of the temporary dressing 1000 or by the contact layer 1006. The contact layer 1006 may comprise any contact layer material known in the art, including, without limitation, non-adherent materials made of perforated or woven polymeric materials, hydrogels, foams (e.g., polyurethane foam dressings), silicones, porous materials permeable to fluids (e.g., hydrophobic silicones containing multiple openings), combinations thereof, or any other suitable contact layer material. The contact layer 1006 may be permeable to fluids originating from the wound (e.g., blood, exudate, etc.) and may also be permeable to gases, allowing at least some circulation of air through the wound.

[0180] In some examples, the contact layer 1006 may be an impermeable barrier that acts primarily to deliver and / or retain the comminuted decellularized fish skin particles 1004 to the wound. Thus, the contact layer 1006 may contain the comminuted decellularized fish skin 1004 or may be configured to retain it at the wound, for example, by acting as a transporter and / or a physical barrier to retain the comminuted decellularized fish skin particles 1004 at or near the wound site.

[0181] The contact layer 1006 may additionally or alternatively be configured to deliver the comminuted decellularized fish skin particles 1004 to the wound via hydrophobic channels defined by and / or therein. Such hydrophobic channels may resist absorption of fluids originating from the wound site, yet provide a medium through which such fluids may be drawn away from the wound.

[0182] The pulverized decellularized fish skin particles 1004 can be associated with the corresponding lumen of the hydrophobic channel in a lyophilized form without adhering or bonding to the channel, and when wound exudate, blood, or other fluids originating from the wound site are drawn into the channel, the pulverized decellularized fish skin particles 1004 can become hydrated and diffuse to the wound surface to provide the benefits discussed above (e.g., hemostasis, analgesic effect, antibacterial effect, barrier function, etc.). In some examples, the pulverized decellularized fish skin particles 1004 can be applied directly to the wound (in dry or hydrated form) in addition to being delivered and retained at the wound site by the contact layer 1006.

[0183] In some examples, the contact layer 1006 can be or include a hydrogel, such as hydrogels known to those skilled in the art. The crushed decellularized fish skin particles 1004 can be associated with a surface of a hydrogel such that the hydrogel acts to deliver and retain the crushed decellularized fish skin particles 1004 at the wound site. Additionally or alternatively, the crushed decellularized fish skin particles 1004 can be incorporated into a hydrogel and released at the wound site. For example, a hydrogel including the crushed decellularized fish skin particles 1004 can be applied to the wound site and upon absorbing water from the wound site, can release the crushed decellularized fish skin particles 1004 associated therewith to diffuse into the wound site. The hydrogel can be further beneficial because it can conform the contact layer 1006 to the wound site as the hydrogel swells.

[0184] The temporary dressing 1000 may optionally include a substrate 1002 associated with or including a contact layer 1006 and configured to contact the wound and / or facilitate contact of the decellularized fish skin particles 1004 with the wound. The temporary dressing 1000 further includes an outer cover 1008 associated with and / or at least partially surrounding the contact layer 1006 and configured to hold the contact layer 1006 at the wound. As depicted in FIG. 10, the outer cover 1008 includes straps 1009 on opposing sides of the outer cover 1008. In one or more embodiments, the straps 1009 of the outer cover 1008 may be wrapped around, adhered to, and / or otherwise associated with the wound site and may be adhered thereto by any means known in the art (e.g., hooks and loops on opposing ends of the straps; an adhesive such as tape, glue, epoxy, cement, etc. applied to one or more straps and attached at or near the wound site or to opposing straps or other portions of the outer cover; tie / fuse straps together; etc.).

[0185] The substrate 1002, if included, may include or be made of the same materials as the contact layer 1006 described above, or any other materials, such as materials that promote wound healing effects through the complex functions of release of therapeutic agents and / or formation of a proper moist environment by preventing the inflow of foreign bodies and / or by releasing and / or storing exudate at the wound site to maintain a proper moist environment. Such substrates include, for example, gels, semi-solids, biocompatible polymers, and / or any combination thereof. In some embodiments, the substrate 1002, outer cover 1008, and / or straps 1009 are stretchable or moldable and configured to conform to and / or firmly compress the wound site.

[0186] In some embodiments, the substrate 1002 includes a biocompatible polymer. Any polymeric material that can be used as a dressing material can be used as the biocompatible polymer without limitation, and can be appropriately selected by one of ordinary skill in the art. The biocompatible polymer can include, for example, one or more of polyvinyl alcohol, polyurethane, polyethylene, polyethylene oxide, low density polyethylene, polyacrylic acid, polyoxyethylene, polytetrafluoroethylene, polypropylene, polyethylene terephthalate, polyamide, polyacrylonitrile, polyester, polyvinyl chloride, polyvinylidene fluoride, polysiloxane (silicone rubber), polyglycolic acid, polylactic acid, polymethacrylic acid, polyacrylamide, polysaccharides, polyvinylpyrrolidone, silicone, alginic acid, sodium alginate, cellulose, pectin, chitin, chitosan, gelatin, collagen, fibrin, hyaluronic acid, natural rubber, synthetic rubber, or combinations thereof.

[0187] The biocompatible polymer may be prepared by assembling the biocompatible polymer into fibers and processing it into a sheet or sheet-like shape, or the fibers of the biocompatible polymer may be processed into a nonwoven or woven fabric. Additionally or alternatively, the biocompatible polymer may be used in the form of a film, foam, hydrocolloid, hydrogel, or may be suitably processed in any other form known to those skilled in the art.

[0188] In one embodiment, the substrate 1002 and / or the biocompatible polymer aid, at least in part, in regulating the moisture content of the wound, as known in the art or as otherwise described herein.

[0189] In one or more embodiments, the temporary dressing 1000 includes one or more therapeutic agents, which in some embodiments may include analgesics, anesthetics, cytokines, growth factors, hemostatic agents, antibiotics, antifungals, hydration compounds, or combinations thereof. The therapeutic agents and / or the pulverized decellularized fish skin may be infused within the substrate 1002 (e.g., within a biocompatible polymer) for time and / or temperature dependent release (e.g., a hydrogel) as known in the art.

[0190] In one or more embodiments, the analgesic agent includes acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, etc. The analgesic agent may additionally or alternatively include agents for treating neuropathic pain, such as, for example, tricyclic antidepressants and anticonvulsants.

[0191] In one or more embodiments, the anesthetic agent comprises an ester-based and / or amide-based local anesthetic. Ester-based local anesthetics include, for example, procaine, amethocaine, cocaine, benzocaine, tetracaine, etc. Amide-based local anesthetics include, for example, lidocaine, prilocaine, bupivacaine, levobupivacaine, ropivacaine, mepivacaine, dibucaine, etidocaine, etc. In one embodiment, amide-based local anesthetics are preferred due to their thermal stability and shelf life (e.g., a shelf life of about 2 years).

[0192] In one or more embodiments, the cytokines include pro-inflammatory cytokines, anti-inflammatory cytokines, and / or combinations of pro-inflammatory and anti-inflammatory cytokines. Anti-inflammatory cytokines include a set of immunomodulatory molecules that can control pro-inflammatory cytokine responses and act in conjunction with certain cytokine inhibitors and soluble cytokine receptors to regulate human immune responses. In embodiments, one or more anti-inflammatory cytokines are used to reduce pain, swelling, and other symptoms of inflammation at the wound site. Exemplary anti-inflammatory cytokines include, for example, interleukin (IL)-1 receptor antagonist, IL-4, IL-6, IL-10, IL-11, and TGF-β.

[0193] In one or more embodiments, the growth factors include transforming growth factor alpha (TGF-α), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), matrix metalloproteinase 2 (MMP-2), MMP-9, and the like.

[0194] In one or more embodiments, the antibiotic comprises one or more antibiotics selected from the following antibiotic classes: Penicillins (e.g. methicillin, amoxicillin, penicillin G, etc.); Tetracyclines (e.g. doxycycline, tetracycline, etc.); · Cephalosporins (e.g. cefdinir, cefepime, ceftriaxone, etc.); ·Quinolones / fluoroquinolones (e.g. ciprofloxacin, levofloxacin, ofloxacin, etc.); · Macrolides (e.g. azithromycin, erythromycin, etc.); · Sulfonamides (e.g. sulfisoxazole, sulfamethoxazole-trimethoprim, etc.); Glycopeptides (e.g. vancomycin, dalbavancin, etc.); Aminoglycosides (e.g. gentamicin, kanamycin, etc.); Lincosamides (e.g., clindamycin, lincomycin, etc.); Nitrofurans (e.g. furazolidone, nitrofurantoin, etc.); · Oxazolidinones (e.g. linezolid, torezolid, etc.); ·Ansamycins (e.g. geldanamycin, rifaximin, etc.); Carbapenems (e.g. meropenem, elapenem, etc.); and Polypeptides (e.g. bacitracin, polymyxin B, etc.).

[0195] In embodiments, the antibiotic comprises a common topical antibiotic cocktail (e.g., bacitracin, neomycin / polymyxin B, neomycin / polymyxin / pramoxine, etc.) The antibiotic may be selected from any other known antibiotic, including, but not limited to, fosfomycin, mupirocin, and chloramphenicol.

[0196] In one or more embodiments, the antifungal agent includes polyene antifungals (e.g., nystatin, amphotericin B, etc.), echinocandins (e.g., micafungin, caspofungin, etc.), azole antifungals (e.g., imidazoles such as bifonazole, sulconazole; triazoles such as epoxiconazole, fluconazole; and thiazoles such as abafungin), allylamines (e.g., butenafine, naftifine, etc.), and combinations thereof.

[0197] In one or more embodiments, the hydrating compounds include materials that absorb and / or store wound exudate, including hydrating gels, oils, or other fluids known in the art. Non-limiting examples include petrolatum, beeswax, panthenol, and the like.

[0198] 11, the temporary dressing may be associated with an outer cover 1108 in the form of a sleeve 1100 including a biocompatible polymer 1102 optionally infused with or otherwise associated with ground decellularized fish skin particles 1104 disposed at the base of the sleeve 1100. The biocompatible polymer 1102 may be ergonomically shaped and / or deformable to accommodate the wound and / or partial or entire limb. The sleeve 1100 may further be associated with a compression device 1110. As depicted in FIG. 11, the compression device 1110 expands the outer cover 1108 such that the expanded sleeve 1100 conforms to the wound and / or limb or partial limb within the sleeve 1100.

[0199] 11, the outer cover 1108 is inflated by filling the bladder 1116 with air, for example from a compressor 1114. Alternatively, the compressor can be filled via the user's mouth or can be a hand pump. The bladder 1116 can alternatively be filled with a fluid (e.g., water).

[0200] The sleeve 1100, and in particular the bladder 1116 of the sleeve 1100, can be vented or the pressure in the bladder 1116 can be adjusted by venting the air or fluid filling the bladder 1116 through the vent hole 1112. In some examples, the vent hole 1112 is manually operated, but in some examples, the vent hole 1112 can have a threshold pressure that automatically vents to maintain the pressure in the bladder 1116 below a given threshold (e.g., 20 psi).

[0201] In one embodiment, the sleeve further comprises one or more straps (not shown) for providing additional fixation force; a deformable material (not shown) within the cavity formed by the opposing layers of the sleeve that deforms when inflated (or partially inflated) and forms a rigid structure when the sleeve is depressurized and / or evacuated, thereby fixing and associating the biocompatible polymer (and the crushed decellularized fish skin particles and / or one or more therapeutic agents) to the wound; a plurality of substrates 1102 (and the crushed decellularized fish skin particles and / or one or more therapeutic agents); and / or a liner (not shown) of the substrate (and the crushed decellularized fish skin particles and / or one or more therapeutic agents).

[0202] In one or more embodiments, the outer cover 1108 is made of and / or includes a flexible material (e.g., thermoplastic elastomers, elastomers, spandex, lycra, polymer aerogel, etc.); adjustable straps (not shown); and / or a heat reflective material configured to retain and / or reflect body heat back to the associated wound and / or nearby tissue / body (e.g., polymer aerogel; radiant barrier fabric; aluminum foil-fabric laminate; metallized thin film-fabric laminate; direct metallized fabric; e.g., quilted or baffle-like fabric; and / or other heat reflective and / or insulating materials known in the art).

[0203] In one or more embodiments, the crushed decellularized fish skin particles 1004, 1104 of the disclosed temporary dressings 1000, 1100 provide one or more beneficial and / or healthy properties. For example, the crushed decellularized fish skin particles may act as a hemostatic agent to reduce and / or stop blood loss from a wound.

[0204] Hemostasis use Decellularized fish skin has significantly better hemostatic properties compared to the coagulants and other biological products tested (Table 1). The hemostatic effect of decellularized fish skin may be due to the collagen content of decellularized fish skin, rather than being directly released by thrombin activation. Platelets in blood may directly bind to collagen using collagen-specific glycoprotein surface receptors. Collagen binding to glycoproteins may initiate a signaling cascade that activates platelet integrins, which among other things mediate the tight binding of platelets to decellularized fish skin. This process results in platelets adhering to the site of injury. The hemostatic properties of decellularized fish skin are promising as a novel hemostatic agent with hemostatic effects derived from other means or in addition to thrombin activation. With its long shelf life and light packaging, the product may be an essential addition to a first aid hemostatic combat kit, independent of the Severely Injured Limb (ACCSIL) device.

[0205] A variety of substances that increase blood clotting have been added to various vehicles with the aim of applying these devices in areas of high bleeding to establish blood clotting in the field of combat. Intuitively, this seems like a good idea to make the blood clot in this way. The substances used are thrombin, a platelet activating factor, and calcium ions (Ca2 + ) containing chitosan that binds Ca2 + is an important factor in thrombus formation. The hemostatic properties of a device designed to cover large or small tissue defects, or tissue destruction incurred by heat off of high velocity munitions, shrapnel, or improvised explosive devices (IEDs), should be present but not likely to cause further harm to the injury site or interfere with future surgery on the wounded area.

[0206] In one embodiment, the Lee White blood clotting test was performed in triplicate to compare the hemostatic effect of wound treatment products such as Oasis® (Smith & Nephew Inc.) and Matristem® (Acell Inc.) to that of decellularized fish skin (Kerecis). This was done at a fixed concentration of 3.5 mg / mL of blood. The total volume was 1 mL of whole blood. 1 NIH unit / mL of thrombin was used as a positive control. Blood without additives was used as a negative control (NEG). The blood clotting time was recorded when the blood was completely clotted. Statistical analysis was performed using the Wilcoxon rank sum test, also known as the Mann-Whitney U test. The results are shown in Table 1 below.

[0207] In another embodiment, the Lee White blood coagulation test was performed on six healthy individuals ranging in age from 23 to 45 years. The hemostatic effects of the following agents in 1 cm2 / 1 mL of blood were tested: Kerecis™ Omega3, Oasis®, Matristem®, and Thrombin (1 NIH unit / mL) for positive control. Blood without additives was used as a negative control (NEG). Blood was collected from participants in an empty vacutainer and the test substance was added. The glass tube was slowly tilted to approximately 45° every 10 seconds, and the time when blood stopped leaking out the side was recorded. The results are illustrated in Table 2 below.

[0208] Decellularized fish skin induced significantly faster clotting than mammalian-derived products (p≦0.0001). Decellularized fish skin also preferred clotting with thrombin, which is used in the natural human hemostatic pathway. Decellularized fish skin showed on average approximately 2 minutes faster clotting than thrombin and approximately 3 minutes faster clotting than mammalian-derived membrane products.

[0209] In some embodiments, the decellularized fish skin can clot blood more than 2 minutes faster than thrombin, more than 1 minute 45 seconds faster than thrombin, more than 1 minute 30 seconds faster than thrombin, more than 1 minute 15 seconds faster than thrombin, more than 1 minute faster than thrombin, more than 45 seconds faster than thrombin, more than 30 seconds faster than thrombin, more than 15 seconds faster than thrombin, or any speed faster than thrombin. In one embodiment, the decellularized fish skin can clot blood at the same speed as thrombin.

[0210] In some embodiments, the faster clotting may be associated with platelets directly binding to glycoprotein surface receptors on the decellularized fish skin, rather than being solely due to thrombin activation, hi another embodiment, the faster clotting may be due to a combination of thrombin activation and platelets directly binding to glycoprotein surface receptors on the decellularized fish skin.

[0211] Tables 1 and 2 below contain the results of one-tailed t-tests (Wilcoxon rank sum test) performed between the various groups of hemostatic agents shown in Figures 1 and 2, respectively.

[0212] [Table 1]

[0213] [Table 2]

[0214] In addition to blood loss, blast injuries often result in complex soft tissue loss, necessitating a targeted approach for optimal outcomes. Key elements include early debridement and application of a cover to promote healing and prevent bacterial contamination and infection. Up to one-quarter of combat-inflicted wounds are infected and require tertiary care, and furthermore, infection following blast-related injury remains a leading cause of morbidity and mortality in injured military personnel. Therefore, improved care closer to the injury site is needed to positively impact the ultimate reconstructive options available after salvage.

[0215] In one or more embodiments, the antiviral and antibacterial properties of the crushed, decellularized fish skin particles act to prevent bacterial and / or viral infection at the wound site, thereby reducing potential complications (e.g., wound infection) and / or increasing the variety of applications and / or environments in which the temporary dressing may be used.

[0216] Additionally, the anti-inflammatory (or inflammation-modulating) properties of the omega-3 PUFAs in the ground, decellularized fish skin may help regulate inflammation at the wound site and, in some embodiments, help stabilize and / or protect the tissue.

[0217] As a non-limiting example, the crushed decellularized fish skin particles can be applied directly to a dirty (e.g., not previously cleansed or debrided) wound in situ (e.g., Echelon I treatment). The crushed decellularized fish skin particles can be used as a stand-alone treatment or as part of a temporary dressing. One or more healthy properties of the crushed decellularized fish skin described above allow for stabilization and / or protection of the underlying wound in some embodiments. It should be understood that once applied to the wound site, the fish skin particles can be subsequently removed by debridement of the associated tissue or washing of the wound site. If the particles remain at the wound site, the decellularized fish skin particles can be safely absorbed by the body without causing an inflammatory response and without the need for subsequent surgical removal.

[0218] As provided above, one beneficial aspect of the crushed decellularized fish skin particles is their ability to stabilize and / or preserve wounds. When applied to wounds, such as dirty, undebrided wounds, on-site (e.g., Echelon I treatment), the treatment allows the wounded person to be transported long distances or for extended periods of time with the wound site preserved. In a similar manner, crushed decellularized fish skin particles can be applied to clean and / or debrided wounds prior to application of a fresh temporary dressing, e.g., during Echelon II treatment, so that the wound can be stabilized and / or preserved while the wounded person is transported elsewhere for additional treatment (e.g., to Echelon III, IV, or V treatment, as appropriate). The healthy properties of crushed decellularized fish skin may also allow for increased incorporation of autografts in some embodiments by better preparing wounds than cadaver skin or other known materials (e.g., during Echelon IV or V treatment).

[0219] The crushed decellularized fish skin particles (or temporary dressings associated therewith) disclosed above may be used to stabilize and / or protect traumatic wounds (e.g., gunshot wounds, puncture wounds, wounds sustained by blast and / or shrapnel, crushed or severed limbs / appendages, etc.), burns, and / or amputations (e.g., those resulting from spontaneous, emergency, or traumatic injuries). The crushed decellularized fish skin particles can impart many salutary effects (e.g., hemostatic effects, antiviral effects, antibacterial effects, inflammatory response modulating effects, etc.), and when used in temporary dressings, the substrate can act to moderate the moisture content / environment of the wound to prevent tissue drying and / or deterioration, while the outer cover provides physical support and, in some embodiments, pressure and / or structure to protect the wound during delivery.

[0220] Kits for wound stabilization and / or protection It should be understood that any of the aforementioned or other temporary dressings (or components thereof) can be included in the kit. For example, as shown in Fig. 12, a kit 1200 for stabilizing and / or protecting a wound can include an outer container 1202 that houses, includes, or contains (i) a contact element 1204 configured to contact the wound, (ii) crushed decellularized fish skin particles 1206 for placement on the wound and held on the wound by the contact element 1204, and (iii) an outer cover 1208 configured to hold the contact element 1204 and the crushed decellularized fish skin particles 1206 at the wound.

[0221] With respect to the contact element 1204, the contact element 1204 can be or include a contact layer 1204 and can include any of the materials or properties discussed above with respect to the contact layer 1204. Alternatively, the contact element 1204 can be a material that acts to hold the crushed decellularized fish skin particles 1206 at the wound. This can include, for example, gauze, a compression sleeve, a bandage, padding, an adhesive wrap, or other wound dressing known in the art.

[0222] Any of the kits or temporary dressings disclosed herein can be used in any number of ways to stabilize and / or protect a wound. FIG. 13 illustrates an exemplary method 1300 for stabilizing and / or protecting a wound. Method 1300 includes applying crushed decellularized fish skin particles to a wound (act 1302) and covering the wound with a contact element configured to hold the crushed decellularized fish skin particles at the wound (act 1304). Variations of method 1300 can further include the act of applying an outer cover to the contact element to secure the contact element to a partial or entire limb that includes the wound. Additionally or alternatively, variations of method 1300 of applying crushed decellularized fish skin to a wound include preserving tissue conditions at or near the wound.

[0223] In an embodiment, the step of applying the comminuted decellularized fish skin particles to the wound (act 1302) includes compressing or packing the comminuted decellularized fish skin particles into a ball or other mass / shape and inserting the ball or mass / shape into the wound. The clinician may form the comminuted decellularized fish skin particles into a ball based on the geometry of the wound, such as a tunneled / eroded wound.

[0224] In some embodiments, the kit (or components thereof) can be used for stabilizing, covering, and / or initiating the wound healing process in tunneling / eroding wounds or other traumatic wounds, including, for example, topical management of bleeding wounds (cuts, lacerations, and abrasions) and / or temporary management of severe bleeding or bleeding wounds.

[0225] In some embodiments, further benefits can be realized through the utilization of systems, kits, and / or methods that incorporate two types of decellularized fish skin products (e.g., sheet-based and crushed, decellularized fish skin) together for processing for complex soft tissue wounds. These two types of fish skin can be used in combination with each other to serve different application purposes. For example, deep, asymmetrical, eroded wounds can be filled with crushed, decellularized fish skin before being secured with a sheet for optimal wound healing, bleeding control, and infection protection during transfer to a higher-level facility. A secondary cover with a sheet-based decellularized fish skin sheet protects the crushed product during dressing changes and adds a bacterial and (additional) hemostatic barrier during transfer. As a result, the injured person can begin healing while awaiting transfer to a medical facility, resulting in a higher quality wound bed for subsequent grafting.

[0226] Additionally, kits with crushed decellularized fish skin can provide one or more surgical benefits including, for example, providing an early treatment approach to wounds that controls bleeding, stabilizes the wound bed, initiates the skin regeneration process, and provides microbial control; simplifying treatment options for tunneled or encroached wounds that are not physically optimized for addressing with conventional materials; filling deep sacral wounds and pressure ulcers, allowing for the application of smaller skin flaps and increasing the chances of flap success; and temporarily immobilizing wounds in preparation for autografts and / or skin flap creation. EXAMPLES

[0227] The following examples as set forth herein are intended for illustrative purposes only and are not intended to limit the scope of the disclosure in any way, as many variations thereof are possible without departing from the spirit and scope of the disclosure.

[0228] Example 1 Wound healing properties of particulate and intact fish skin sheets in a splint excision mouse model to investigate the ability of particulate fish skin to induce granulation and promote healing of deep subcutaneous splint excision wounds in mice Wound healing in mice is fundamentally different from human wound healing because it is driven primarily by contraction. By splinting the wound, the repair process then relies on epithelialization, cell proliferation, and angiogenesis, which closely mirror the biological processes of human wound healing.

[0229] Use male C57BL / 6. Each mouse receives two wounds, allowing the application of both decellularized fish skin and standard care (positive control) on the same animal, so that each animal serves as its own control. After carefully shaving and depilating the back of the mouse, use a sterile 4 mm biopsy to punch out the outline of two circular patterns for the wound on one side of the mouse's midline at shoulder level. Use serrated forceps to lift the skin in the middle of the outline and use iris scissors to create a full-thickness wound extending through the subcutaneous tissue, including the fleshy fat layer (panniculus carnosus), and excise the circular tissue piece.

[0230] The process is repeated for the wound on the other side of the midline.

[0231] The silicone "donut" splint is 10 mm in diameter, 0.5 mm thick, and has a 5 mm hole in the center. The plastic protective coating is removed from each side of the silicone splint. Cyanoacrylate adhesive is applied to one side of the silicone splint. The splint is centered over the wound and secured with interrupted 6-0 nylon sutures to ensure proper positioning. A ruler is placed under the splint and a photograph is taken using a macro lens.

[0232] Mice are divided into two groups at three time points: D7, D14, and D21: Group A: Only the treatment wound is filled with ground decellularized fish skin, the control wound is not filled, and then both wounds are covered with a transparent occlusive dressing (such as OpSite). Group B: Only the treatment wounds are filled with small pieces of intact decellularized fish skin, while the control wounds are not filled. Both wounds are then covered with a transparent occlusive dressing (such as OpSite).

[0233] Anesthesia and analgesia are provided according to the animal facility's recommendations, with the exception that NSAIDs are not used as they may inhibit inflammation and affect the healing process.

[0234] Visually inspect the wounds once or twice a week and after each dressing change, photograph them, and measure their size. If the fishskin is incorporated into the wound bed within a week and there is still space available, insert an additional fishskin, mimicking clinical practice.

[0235] Groups are sacrificed on days 3, 7, 14, and 21 and mice are followed up for up to 21 days. After euthanasia by cervical dislocation, the splints are removed and a large complete excision is made around and under the wound area. Tissues are incubated for further diagnosis by histology to examine inflammation, granulation, and quality of healing.

[0236] qPCR is performed to quantify the expression of relevant wound healing related genes (e.g., VEGF, IL-1b, eNOS, iNOS). Wound size is determined using ImageJ or other equivalent software.

[0237] Treatment and control wounds are randomized on the right or left side of the mice.

[0238] Parametric and non-parametric statistical analyses are performed as appropriate. Statistical analyses are also performed on LDI data and other bioassay results. Paired Student's t-tests and ANOVA F-tests are performed to assess the significance of histological differences between treatment groups. Post-hoc analyses are adjusted for multiple testing. P-values ​​<0.05 are considered significant.

[0239] Results include that wounds treated with pulverized decellularized fish skin are stabilized and that wound healing initiation at the wound site is faster and / or more robust than controls, showing reduced inflammation at the wound site, and increased abundance, concentration, and / or half-life of healing-related transcripts.

[0240] Results also included that wounds treated with the sheet-based decellularized fish skin had a lower incidence of infection, a greater amount of cellular ingrowth (granulation tissue formation), reduced inflammation, and improved quality of healing compared to controls.

[0241] Example 2 Hemostatic properties of decellularized fish skin in a porcine femoral artery bleeding model to investigate the hemostatic properties of decellularized fish skin The objective of this study is to demonstrate that particulate fish skin is more effective at controlling bleeding than a control (standard of care) product without obvious side effects. A 50% reduction in blood loss after treatment is considered clinically significant.

[0242] In all surgical procedures, supportive measures such as anesthesia, analgesia, maintenance of fluids, and warming are used to maintain physiological homeostasis (unless contraindicated by the experimental protocol) and minimize pain and distress in the animals. After anesthesia, Yorkshire cross male pigs weighing 34 kg to 44 kg are intubated and a catheter is inserted into the ear vein to administer maintenance fluids.

[0243] [Table 3]

[0244] An arterial bleeding model is used.

[0245] The right carotid artery is cannulated and connected to a transducer to record blood pressure, and the right jugular vein is catheterized to administer resuscitation fluids during bleeding and wound management.

[0246] A midline laparotomy and cystostomy are performed and the abdomen is closed by suturing and stapling the skin. A 10 cm incision is made in the groin area close to the femoral artery and 5 cm of the artery is separated from the surrounding tissue by cauterization and ligation of the small arterial branches.

[0247] The artery is immersed in 2% lidocaine and diluted to its normal diameter. Fluid maintenance is then discontinued. After a stabilization period of 5-10 min, clamp the artery proximally and distally and make an arteriotomy 6 mm in diameter on the anterior surface of the vessel, 2-3 cm from the bottom of the groin. Release the clamps and allow to bleed freely for 45 s. Blood is collected by aspiration.

[0248] Divide the pigs into four groups: (a) crushed decellularized fish skin, (b) sheet-based decellularized fish skin, (c) standard of care (i.e., combat gauze, positive control), and (d) regular treatment gauze (negative control).

[0249] Immediately after free bleeding begins, each product is opened and packed into the wound and covered with an open sponge or gauze: (a) crushed decellularized fish skin compressed to form an adherent ball; using a sponge to stabilize the product in place and press it against the wound; (b) a 3 × 7 cm decellularized fish skin sheet folded in half and pressed against the wound by a sponge; (c) using a sponge to press combat gauze against the wound; (d) using a sponge to press regular gauze against the wound.

[0250] Manual pressure is maintained for 3 minutes to stop the bleeding. The skin flap is pulled over the sponge without clamping the test material or applying additional pressure. Fluid resuscitation is then initiated and 500 mL of Hextend (6% HES + glucose in a balanced electrolyte solution) is infused via the jugular catheter to raise and maintain the MAP between 60 mm Hg and 65 mm Hg. Fluid resuscitation is then continued with up to 10 L of LR solution. After compression, pressure is slowly released and hemostasis is observed for 3 minutes. If no bleeding is observed during this period, initial hemostasis is considered to have been achieved. All shed blood is continuously collected and the time to hemostasis is recorded. Blood loss is calculated and reported as post-procedure blood loss. The pig is monitored for up to 2.5 hours or until death. Time of survival is recorded and a final blood sample is collected. Surviving pigs may be scanned by CT. The legs of surviving pigs are flexed to test the stability of hemostasis. The product is removed and examined for clot status and vascular patency. Animals are euthanized intravenously according to institutional standards and tissue samples are collected for histology. Gross necropsy is performed on vital organs. Tissue slides are prepared for H&E staining.

[0251] The primary endpoints measured are post-procedure blood loss, bleeding / hemostasis time (time required for bleeding to stop), MAP, survival time, and survival rate. Secondary endpoints include hemoglobin, hematocrit, platelet count, pH, lactate, base deficit, and coagulation values ​​(e.g., PT, aPTT, fibrinogen, and TEG parameters).

[0252] Statistical analysis of LDI data and other bioassay results will be performed as appropriate. ANOVA F-tests will be performed to assess the significance of differences between treatment groups in % re-epithelialization and molecular changes over time. Post-hoc analyses will be performed using Tukey's honestly significant difference (HSD) to adjust for multiple testing. Non-parametric tests will be incorporated as appropriate for the corresponding data. P-values ​​<0.05 will be considered significant.

[0253] The results include at least a 50% reduction in blood loss after treatment in groups (a) and (b), with group (a) showing the greatest reduction in blood loss after treatment and the highest survival rate.

[0254] Example 3 To investigate the ability of pulverized decellularized fish skin to induce granulation tissue formation in deeply eroded subcutaneous excision wounds in pigs and / or to reduce time and provide a more favorable wound bed for Split Thickness Skin Grafts. One approach following debridement is negative pressure wound therapy (NPWT), which promotes blood flow to the wound, controls edema, and reduces the presence of proteases, thus leading to increased granulation and revascularization of the wound bed. NPWT is a traditional treatment for complex wound healing, but is not always practical for use during prolonged in-situ care and rapid evacuation to higher levels of care. Notable drawbacks of NPWT include, for example, the inability to precisely control the pressure applied in geometrically challenging wounds, or wounds near or in anatomically sensitive areas where an adhesive seal is difficult to obtain, bleeding (which may be difficult to assess due to occlusion from the dressing), skin irritation, infection, pain or discomfort, ingrowth of granulation tissue into the dressing material, as well as machine- or device-related technical challenges. For example, some NPWT machines are not ambulatory, rely on electricity, and need to be continuously proximal to the patient to be effective, further complicating patient mobility during retrieval.

[0255] Use young, castrated male Duroc pigs to minimize potential interference from the estrogen cycle and reduce aggressiveness of the animals. In all surgical procedures, use ancillary measures such as anesthesia, analgesia, maintenance of fluids, and warming to maintain physiological homeostasis (unless contraindicated by the experimental protocol) and minimize pain and distress of the animals.

[0256] Each animal receives four wounds, two on each side of the flank. Wounds are created by a combination of sharp and blunt dissection within a circumference of approximately 3 inches by 3 inches. Depth extends to subcutaneous tissue, fascia, and muscle, with some erosion. Wounds are treated within 1 hour of wound creation with the following: (a) SOC dressing (e.g., saline, sulfamylone, or similar [wet to dry]); (b) crushed decellularized fish skin with a sheet of decellularized fish skin as a cover, secured with sutures or staples; (c) NPWT with a VAC device (KCI); (d) crushed decellularized fish skin with a sheet of decellularized fish skin as a cover, intact fish skin cover and NPWT.

[0257] The wound is then covered with a non-adherent dressing and secured with a neoprene garment. The animals are recovered and monitored.

[0258] Remove the garment and dressing and inspect the wound to assess its readiness for grafting at 2 / 3, 4 / 5, 6 / 7 days after injury, or every 2-3 days, until the wound is deemed ready for grafting by an experienced clinician. Once ready, the wound is grafted with a 3:1 or 4:1 wide mesh autograft and dressed.

[0259] After grafting, inspect the wounds 1-2 times weekly to monitor graft harvest and healing until at least day 28, or until re-epithelialization if earlier. At the end of the study, euthanize the animals according to the institution's standard and recommended procedures.

[0260] At all of the above time points in the experiment, wounds were photographed and imaged using laser Doppler imaging (LDI) to assess perfusion, swabbed for subsequent culture / microbiome analysis, and two punch biopsies were taken (2 mm each), one preserved in formalin and the other preserved in AllProtect reagent (or flash frozen).

[0261] The endpoints were: (a) the number of days post-injury when the wound bed was ready for grafting; (b) graft harvest at days 5-7 post-grafting; (c) time to complete epithelialization; (d) scarring / aesthetic appearance of healed wounds 4 weeks after grafting; (e) histological analysis to examine inflammation, re-epithelialization, and ECM (H&E and Masson's Trichrome); (f) expression of selected wound-healing-related genes, quantified by mRNA analysis.

[0262] Pigs will be followed for 6-8 weeks after implantation, resulting in a total study period of 9-12 weeks.

[0263] Clinical assessors will be blinded.

[0264] ANOVA F-tests are performed to assess the significance of differences between treatment groups in % re-epithelialization and molecular changes over time. Post-hoc analyses are performed using Tukey's honestly significant difference (HSD) to adjust for multiple testing. Non-parametric tests are incorporated accordingly for corresponding data. P-values ​​<0.05 are considered significant.

[0265] Results include groups (b) and (d) being superior to the positive control (group (a)) and at least as good as, or in some cases significantly better than, group (c).

[0266] 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.

[0267] As used herein, the term "substrate" may include any material known in the art that may act as a vehicle for a therapeutic agent and, additionally or alternatively, allow for and / or passively regulate moisture at and / or around the wound.

[0268] 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.

[0269] The degree of "echelon" as used herein 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, with Echelon II care 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 permanent regional hospitals. Echelon V refers to injuries and / or procedures requiring extensive rehabilitation and convalescent care, with Echelon V care 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.

[0270] The term "wound" as used herein is intended to encompass tissue injury 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, while 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.

[0271] 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, lesions, 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 injury 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, pressure sores, and other causes.

[0272] Furthermore, wounds that are amenable to treatment by the wound treatment and methods disclosed herein include injuries that can be located at any site, including internal, interface, external, interstitial, external, and / or internal. Examples of wounds that are suitable for covering with scaffolding include incisions, deep wounds, open wounds, tissue tears, bedsores, dermatitis, lesions, chronic wounds, battlefield wounds, necrotic wounds, acute, chronic, traumatic, lacerations, abrasions, contusions, necrotizing facial inflammation, toxic epidermal neurolysis, bed sores, venous insufficiency ulcers, arterial ulcers, diabetic or neuropathic ulcers, pressure ulcers, mixed ulcers, burns, mucormycosis, vascular wounds, pyoderma, gangrene, and equivalent conditions, and / or combinations thereof, as known by those skilled in the art. Treatment of wounds in human and animal subjects is contemplated.

[0273] In certain embodiments, the wound treatments and methods disclosed herein are used to reconstruct the abdominal wall, for example to repair a hernia. For example, in repairing a hernia, the surgeon makes an incision near the location of the hernia. For an inguinal hernia, the incision is made just above the crease where the abdomen meets the thigh. To repair an umbilical hernia, the incision is made near the navel, and if the umbilical hernia occurred at the site of a previous surgery, the incision from that surgery is reopened. Regardless of where the incision is made, the surgery proceeds in much the same way. The hernia sac is carefully opened and the intestines or other tissues are placed back into the abdomen. Weakened areas are repaired and strengthened with a synthetic mesh or sutures that draw the abdominal muscle tissue together.

[0274] 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.

[0275] By providing a wound treatment in accordance with the disclosed embodiments, the challenges of existing wound treatments, which are susceptible to shear forces and difficult to apply to complex wound geometries, are advantageously addressed. Embodiments of the disclosed wound treatments and methods enable clinicians to provide a scaffolding material that better conforms to the wound bed and, in embodiments, promotes cellular ingrowth and vascularization while working with a sheet of scaffolding material to provide a substrate for wound healing.

[0276] 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 are mutually exclusive.

[0277] In addition to the above, further embodiments and examples include: 1. A wound treatment comprising particles of decellularized fish skin, wherein a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a range between a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound. 2. The wound treatment of claim 1, wherein the predetermined size threshold is greater than 1 mm. 3. The wound treatment of claim 1, wherein the predetermined size threshold is between 1 mm and 1.39 mm, or the predetermined size threshold is between 1.4 mm and 1.99 mm. 4. The wound treatment of claim 1, wherein the predetermined size threshold is between 2 mm and 2.8 mm. 5. The wound treatment according to claim 1, wherein the predetermined size threshold is less than 1 mm. 6. The wound treatment according to claim 1, wherein the predetermined size threshold is between 1 mm and 2 mm. 7. The wound treatment according to claim 1, wherein the predetermined size threshold is greater than 2 mm. 8. Wound treatment according to any one or combination of 1 to 7 above or 9 to 10 below, wherein the predetermined size threshold is related to the length and / or width of the particles of decellularized fish skin, or the predetermined size threshold is not related to the thickness of the particles of decellularized fish skin. 9. Treating a wound by any one or combination of 1 to 8 above or 10 below, wherein a predetermined percentage of particles of decellularized fish skin having a maximum dimension within a predetermined size threshold and a minimum size threshold is 75% or more of the particles. 10. Wound treatment according to any or a combination of 1 to 9 above, wherein a second predetermined percentage of at least a second portion of the particles of decellularized fish skin has a maximum dimension within a range of a second predetermined size threshold maximum and / or a second size threshold minimum, and the second predetermined size threshold maximum is different from the predetermined size threshold maximum of the first portion of the particles and / or the second size threshold minimum is different from the size threshold minimum of the first portion of the particles. 11. A method of treating a wound, the method comprising the steps of providing particles of decellularized fish skin, wherein a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound; applying the particles of decellularized fish skin to a wound bed; and covering the wound bed with a dressing. 12. A method of wound treatment according to any one or combination of 11 above or 13 to 22 below, further comprising the steps of preparing the wound bed for treatment prior to applying the particles of decellularized fish skin to the wound bed, securing the dressing, and checking the wound bed for incorporation of the particles of chopped, decellularized fish skin. 13. A method of treating a wound according to any one or combination of 11-12 above or 14-22 below, wherein the dressing is a non-adherent dressing including a synthetic non-woven dressing or a woven cotton dressing. 14. A method for treating a wound according to any one or combination of 11 to 13 above or 15 to 22 below, wherein the step of applying particles of decellularized fish skin to the wound bed comprises conforming the particles of decellularized fish skin to the shape of the wound bed. 15. A method for treating a wound according to any one or combination of 11 to 14 above or 15 to 22 below, further comprising the step of wetting the decellularized fish skin particles with a liquid prior to application to obtain moist particles of decellularized fish skin. 16. A wound treatment method according to any one or combination of 12, wherein the step of checking the wound bed for integration is performed within two weeks of application of the particles of decellularized fish skin to the wound bed. 17. A method for treating a wound according to any one or combination of 11 to 16 above or 18 to 22 below, wherein the particles of decellularized fish skin define a substantially rectangular shape. 18. A method for treating a wound according to any one or combination of 11 to 17 or 19 to 22 below, wherein particles of decellularized fish skin are provided in a package configured to receive a liquid. 19. A wound treatment method according to any one or combination of 15, wherein moist particles of decellularized fish skin are formed into a paste prior to application to the wound bed. 20. A method for wound treatment according to any one or combination of 11 to 19 above or 21 to 22 below, in which particles of decellularized fish skin are used in combination with a sheet-based decellularized fish skin scaffold. 21. A method of wound treatment according to any one or combination of 11 to 20 or 22 below, wherein a sheet-based decellularized fish skin scaffold is applied onto the wound bed after application of particles of decellularized fish skin. 22. A wound treatment method according to any one or combination of 11 to 21, wherein the step of providing particles of decellularized fish skin comprises providing a second predetermined percentage of at least a second portion of the particles of decellularized fish skin having a maximum dimension within a range of a second predetermined size threshold maximum and / or a second size threshold minimum, and the second predetermined size threshold maximum is different from the predetermined size threshold maximum of a first portion of the particles and / or the second size threshold minimum is different from the size threshold minimum of the first portion of the particles. 23. A method of providing a wound treatment, the method comprising the steps of providing one or more sheets of decellularized fish skin, and chopping or grinding the one or more sheets of decellularized fish skin into particles of decellularized fish skin such that a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a predetermined size threshold maximum and a size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound.

[0278] In addition to the above, further embodiments and examples include: 1. A temporary wound treatment containing crushed decellularized fish skin in particulate form. 2. A temporary wound treatment according to any one or combination of 1 above or 2 to 12 below, wherein the temporary wound treatment comprises a temporary dressing configured and arranged to deliver crushed decellularized fish skin in particulate form to the wound, the temporary dressing comprising a contact layer configured to contact the wound and hold the crushed decellularized fish skin at the wound, and an outer cover associated with the contact layer, the outer cover configured to hold the contact layer at the wound. 3. A temporary wound treatment according to 2 above, further comprising a substrate carrying associated and crushed decellularized fish skin particles in a contact layer. 4. Temporary wound treatment according to 3 above, wherein the crushed decellularized fish skin particles are releasable from the substrate in a time-dependent manner. 5. Temporary wound treatment according to any one or combination of 1 to 4 above or 6 to 12 below, wherein the crushed decellularized fish skin particles are smaller than about 1 cm in diameter, smaller than about 0.1 cm in diameter, smaller than about 10 mm in diameter, smaller than about 1 mm in diameter, smaller than about 0.1 mm in diameter, smaller than about 10 μm in diameter, smaller than about 1 μm in diameter, or a combination thereof. 6. A temporary wound treatment according to any one or combination of 1-5 or 7-12 below, further comprising a compression element associated with the outer cover, the compression element configured to conform the outer cover to the shape of the wound. 7. A temporary wound treatment according to any one or combination of 1-5 or 8-12 below, further comprising a compression element associated with the outer cover, the compression element configured to conform the outer cover to the shape of the partial or entire limb including the wound. 8. A temporary wound treatment according to any one or combination of 6-7 above, wherein the compression element comprises a sleeve having an inflatable bladder. 9. The temporary wound treatment according to 8, further comprising a substrate associated with the contact layer and carrying crushed decellularized fish skin particles, the substrate being disposed at the bottom or peripheral wall of the sleeve. 10. Temporary wound treatment according to any one or combination of 1-9 above or 11-12 below, wherein the crushed decellularized fish skin particles comprise partially processed, crushed decellularized fish skin particles. 11. A temporary wound treatment according to 10 above, wherein the partially processed, ground decellularized fish skin particles have been treated with one or more enzymes to reduce the stiffness of the ground decellularized fish skin particles. 12. A temporary wound treatment according to claim 11, wherein at least a portion of the extracellular matrix material within the partially processed, crushed decellularized fish skin particles has been cleaved by one or more enzymes, increasing one or more of the ductility or elasticity of the partially processed, crushed decellularized fish skin particles. 13. A kit for stabilizing and / or protecting a wound, comprising a container containing comminuted decellularized fish skin in particulate form for placement on or within the wound and to be held at the wound site by a contact element. 14. A kit according to any one or combination of 13 above or 15 to 18 below, wherein the container further comprises a contact element configured to contact the wound and hold the pulverized decellularized fish skin in particulate form at the wound, and an outer cover configured to hold the contact element and the pulverized decellularized fish skin in particulate form at the wound. 15. A kit according to any one or combination of 13-14 or 16-18 below, wherein the container further comprises a substrate for carrying the pulverized decellularized fish skin in particulate form and associating it with the contact element. 16. A kit according to any one or combination of 13-15 or 17-18 below, wherein the container further comprises one or more therapeutic agents, including an analgesic, an anesthetic, a cytokine, a growth factor, a hemostatic agent, an antibiotic, an antifungal agent, a hydrating compound, or a combination thereof. 17. A kit according to any one or combination of 13 to 16 or 18 below, wherein the crushed decellularized fish skin particles are smaller than about 1 cm in diameter, smaller than about 0.1 cm in diameter, smaller than about 10 mm in diameter, smaller than about 1 mm in diameter, smaller than about 0.1 mm in diameter, smaller than about 10 μm in diameter, smaller than about 1 μm in diameter, or a combination thereof. 18. A kit according to any one or combination of 14-17 above, wherein the container further includes a compression element associated with the outer cover, the compression element conforming the outer cover to the wound and / or to the shape of the partial or entire limb including the wound when the compression element is associated with the outer cover. 19. A method for stabilizing and / or protecting a wound, the method comprising the step of applying comminuted decellularized fish skin in particulate form to the wound. 20. The method according to any one or combination of 19 or 21 to 28 below, wherein the method further comprises covering the wound with a contact element configured to hold the crushed decellularized fish skin particles at the wound, and applying an outer covering to the contact element to secure the contact element to the partial or entire limb containing the wound. 21. A method according to any one or combination of 19-20 or 22-28 below, wherein the step of applying pulverized decellularized fish skin to the wound includes preserving tissue condition at or near the wound. 22. The method according to claim 21, wherein preserving tissue condition at or near the wound includes one or more of reducing loss of damaged tissue or increasing the likelihood that the affected area, including the wound, can recover. 23. The method according to 21 or 22 above, wherein preserving tissue condition at or near the wound comprises regulating the moisture content of the wound to prevent the wound from drying out or causing deterioration of the tissue at or near the wound. 24. A method according to any one or combination of 19 to 23 above or 25 to 28 below, wherein the step of applying decellularized fish skin to a wound comprises enhancing hemostasis at the wound. 25. A method according to any one or combination of 19 to 24 or 26 to 28 below, wherein the step of applying decellularized fish skin to a wound includes reducing pain associated with the wound. 26. A method according to any one or combination of 19 to 25 above or 27 to 28 below, wherein the step of applying the crushed decellularized fish skin particles to the wound comprises compressing the crushed decellularized fish skin particles into a ball and inserting the ball into the wound. 27. Any or a combination of methods according to 19 to 26 above or 28 below, wherein the wound is a deep tissue wound, a bleeding wound, or a wound exposing bone and / or tendon. 28. A method according to any one or combination of 19 to 27 above, wherein the step of applying the comminuted decellularized fish skin in particulate form to the wound does not promote ingrowth of cellular tissue within the decellularized fish skin in particulate form. 29. A bandage for the treatment of uncleaned and / or undebrided wound sites, the bandage comprising comminuted decellularized fish skin in particulate form. 30. A dressing according to any one or combination of 29 above or 31 to 33 below, wherein the comminuted decellularized fish skin in particulate form is rehydrated prior to application to the wound site. 31. A dressing according to any one or combination of 29-30 or 32-33 below, further comprising a covering for fixing the comminuted decellularized fish skin in particulate form to the wound site. 32. A dressing according to any one or combination of 29 to 31 or 33 below, wherein the wound site includes a deep wound and crushed fish skin in particulate form is compressed into the deep wound. 33. A temporary wound treatment according to any one or combination of 1 to 12 above, wherein the particle morphology is configured to minimize cell scaffold formation at the wound site during the temporary wound treatment.

[0279] 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.

[0280] 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.

[0281] Also, 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.

[0282] 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 wound treatment and the method for making the wound treatment 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.

[0283] Those skilled in the art will recognize the interchangeability of the various disclosed features. In addition to the variations described herein, those skilled in the art will be able to mix and match other known equivalents to each feature to utilize methods of preparing wound treatments and making wound treatments based on the principles of the present disclosure. Those skilled in the art will understand that the features described herein may be applied to other types of wound treatments and medical applications generally.

[0284] While the present disclosure describes certain exemplary embodiments and examples of wound treatment, it will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed wound treatment 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. A composition for treating wounds, comprising: the composition comprises particles of decellularized fish skin; A predetermined percentage of at least a first portion of the particles of decellularized fish skin is a maximum value of a predetermined size threshold; a predetermined size threshold minimum that is effective in preserving the matrix structure of the decellularized fish skin and promoting regenerative ingrowth of cells into the wound; and having a maximum dimension in the range of A wound treatment composition, wherein the decellularized fish skin particles are in dry, wet or paste form, and are placed on or incorporated into a hydrogel, or placed on or injected into a substrate.

2. 2. The wound treatment composition of claim 1, wherein the maximum predetermined size threshold is greater than 1 mm.

3. the maximum predetermined size threshold is between 1 mm and 1.39 mm; or The predetermined size threshold maximum is between 1.4 mm and 1.99 mm.

2. The wound treatment composition of claim 1.

4. 2. The wound treatment composition of claim 1, wherein said predetermined maximum size threshold is between 2 mm and 2.8 mm.

5. 2. The wound treatment composition of claim 1, wherein the maximum predetermined size threshold is less than 1 mm.

6. 2. The wound treatment composition of claim 1, wherein said predetermined maximum size threshold is between 1 mm and 2 mm.

7. 2. The wound treatment composition of claim 1, wherein the maximum predetermined size threshold is greater than 2 mm.

8. 2. The wound treatment composition according to claim 1, wherein the maximum value of the predetermined size threshold is related to the length and / or width of the decellularized fish skin particles, or the maximum value of the predetermined size threshold is not related to the thickness of the decellularized fish skin particles.

9. 2. The wound treatment composition of claim 1, wherein the predetermined percentage of particles of decellularized fish skin having the maximum dimension within the range between the maximum value of the predetermined size threshold and the minimum value of the predetermined size threshold is 75% or more of the particles.

10. a second predetermined percentage of at least a second portion of the particles of decellularized fish skin; have a maximum dimension within a second predetermined size threshold maximum and / or a second predetermined size threshold minimum; and 2. The wound treatment composition of claim 1, wherein the maximum value of the second predetermined size threshold is different from the maximum value of the predetermined size threshold of the first portion of the particles, and / or the minimum value of the second predetermined size threshold is different from the minimum value of the predetermined size threshold of the first portion of the particles.

11. particles of decellularized fish skin, wherein a predetermined percentage of at least a first portion of the particles of decellularized fish skin have a maximum dimension within a range between a predetermined maximum size threshold and a predetermined minimum size threshold effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound, the particles of decellularized fish skin being in a dry form, a wet form, or a paste form, and being disposed on or incorporated into a hydrogel, or being disposed on or injected into a substrate; a dressing for covering a wound bed to which the decellularized fish skin particles have been applied; A wound treatment kit comprising:

12. 12. The wound treatment kit of claim 11, wherein the dressing is a non-adherent dressing including a synthetic non-woven dressing or a woven cotton dressing.

13. The wound treatment kit according to claim 11 , wherein the particles of decellularized fish skin are applied to the wound bed so as to conform to the shape of the wound bed.

14. The wound treatment kit according to claim 11, wherein the particles of decellularized fish skin are moistened with a liquid before application and are configured to be applied as moist particles of decellularized fish skin.

15. 12. The wound treatment kit of claim 11, wherein the wound bed is checked for fusion of the decellularized fish skin particles within two weeks of application of the decellularized fish skin particles to the wound bed.

16. The wound treatment kit of claim 11 , wherein the particles of decellularized fish skin define a substantially rectangular shape.

17. 12. The wound treatment kit of claim 11, comprising a package for the particles of decellularized fish skin configured to receive a liquid.

18. 15. The wound treatment kit of claim 14, wherein the moist particles of decellularized fish skin are formed into a paste prior to application to the wound bed.

19. The wound treatment kit of claim 11, further comprising a sheet-based decellularized fish skin scaffold.

20. 20. The wound treatment kit of claim 19, wherein the sheet-based decellularized fish skin scaffold is for application onto the wound bed after application of the particles of decellularized fish skin.

21. the decellularized fish skin particles comprise a second predetermined percentage of at least a second portion of the decellularized fish skin particles, the particles having a maximum dimension within a second predetermined size threshold maximum and / or a second predetermined size threshold minimum; and the second predetermined size threshold maximum is different from the predetermined size threshold maximum of the first portion of the particles, and / or the second predetermined size threshold minimum is different from the predetermined size threshold minimum of the first portion of the particles; The wound treatment kit according to claim 11.

22. 1. A method of making a wound treatment composition, said method comprising: Providing one or more sheets of decellularized fish skin; A predetermined percentage of at least a first portion of the particles of decellularized fish skin is a maximum value of a predetermined size threshold; a predetermined size threshold minimum that is effective to preserve the matrix structure of the decellularized fish skin and promote regenerative ingrowth of cells into the wound; shredding or pulverizing the one or more sheets of decellularized fish skin into particles of decellularized fish skin having a maximum dimension within the range of Including, The decellularized fish skin particles are in dry, wet or paste form and are placed on or incorporated into a hydrogel, or placed on or injected into a substrate.

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