WO2020261187A1

WO2020261187A1 - Multi-layered biopolymer film dressing to combat wound biofilm

Info

Publication number: WO2020261187A1
Application number: PCT/IB2020/056041
Authority: WO
Inventors: Carrina WARD; Alexander WAITE; Katie BOURDILLON
Original assignee: Systagenix Wound Management, Limited
Priority date: 2019-06-28
Filing date: 2020-06-25
Publication date: 2020-12-30

Abstract

The present disclosure relates generally to wound dressing compositions that may be used to disrupt and/or prevent biofilm formation in a wound upon application. The wound dressing can contain bioresorbable polymers. The wound dressing composition includes an internal layer that includes citric acid and/or sodium bicarbonate. The internal layer may include a biopolymer, such as a mixture of a collagen, an oxidized regenerated cellulose (ORC), and citric acid. Also disclosed herein are kits including the wound dressing compositions of the present technology, and instructions for use.

Description

MUUTI-UAYERED BIOPOUYMER FIUM DRESSING TO COMBAT WOUND BIOFIUM

TECHNICAU FIEUD

[0001] The present technology relates generally to wound dressing compositions with an internal layer that includes citric acid and/or sodium bicarbonate, where the internal layer may include a biopolymer, such as a mixture of a collagen and an oxidized regenerated cellulose (ORC). Such wound dressing compositions may be used to disrupt and/or prevent biofilm formation in a wound upon application.

BACKGROUND

[0002] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0003] Infections can retard wound healing and, if untreated, can result in tissue loss, systemic infections, septic shock, and death. Moreover, in addition to vegetative or free-floating bacteria present in a wound, bacterial biofdms may also form in a wound presenting further challenges in wound therapy, particularly chronic wounds. A biofilm is an association of microorganisms that can adhere to a surface forming three-dimensional microbial communities. The ability of bacteria to form these complex biofilms can impede a host’s defense mechanisms against pathogens.

SUMMARY

[0004] In an aspect, a wound dressing composition is provided that includes a first layer, a second layer, and a third layer. The first layer and third layer include a biopolymer. The second layer includes about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent that includes citric acid, sodium bicarbonate, or a combination thereof, and optionally a biopolymer (e.g., a mixture of collagen and oxidized regenerated cellulose (ORC)). In the wound dressing composition, the first layer and the third layer do not include citric acid (or do not include sodium bicarbonate, when sodium bicarbonate is present in the second layer), and the first layer and the third layer encapsulate the second layer.

[0005] In an aspect, a wound dressing composition is provided that includes a first layer, a second layer, and a third layer. In any embodiment herein, the first layer includes a biopolymer and a surface including a peripheral zone facing the third layer (“the first layer periphery”). The second layer includes a mixture of a collagen, an oxidized regenerated cellulose (ORC), and about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent that includes citric acid and/or sodium bicarbonate. The third layer includes a biopolymer and a surface that includes a peripheral zone facing the first layer (“the third layer periphery”). In the wound dressing composition, the first layer and the third layer do not include citric acid and/or sodium bicarbonate, and the first layer periphery and the third layer periphery are adjoined to form a seal.

[0006] In a related aspect, a method for treating a subject with a wound is provided, where the method includes administering to the wound a wound dressing composition of any embodiment disclosed herein. [0007] In a further related aspect, a kit is provided that includes a wound dressing compositions of any embodiment described herein as well as instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows a perspective view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology. [0009] FIG. 2 shows a cross-sectional view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology.

[0010] FIG. 3 shows a cross-sectional view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology, where the first layer 320 and third layer 310 form a continuous outer layer encapsulating the second layer 330. [0011] FIG. 4 shows a cross-sectional view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology, where the first layer 420, third layer 410, and second layer 430 form a continuous biopolymer matrix where the first antimicrobial agent is entirely concentrated within the second layer 430.

[0012] It should be noted that the representative illustrations provided in the figures set forth herein is intended to illustrate the general features and/or characteristics of exemplary embodiments to aid in describing the present technology in full. The figures may not precisely reflect the characteristics of any given embodiment, and are not necessarily intended to define or limit the scope of the claimed subject matter. Further, the present technology may or may not include or incorporate therewith any one or more features of characteristics set provided in any one or more figures. DETAILED DESCRIPTION

[0013] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

Definitions

[0014] The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

[0015] The following terms are used throughout as defined below.

[0016] As used herein and in the appended claims, singular articles such as“a”,“an”, and“the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0017] As used herein,“about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term - for example,“about 10 wt.%” would mean “9 wt.% to 11 wt.%.” It is to be understood that when“about” precedes a term, the term is to be construed as disclosing“about” the term as well as the term without modification by“about” - for example,“about 10 wt.%” discloses“9 wt.% to 11 wt.%” as well as discloses“10 wt.%.”

[0018] The term“mammalian recombinant collagen” refers to collagen manufactured by culturing a non-human organism or mammalian or non-mammalian cells to express at least one exogenous gene encoding a collagen in the culturing system. The term“human recombinant collagen” refers to collagen manufactured by culturing a non-human organism or mammalian or non-mammalian cells to express at least one human gene encoding a collagen. The human recombinant collagen may be selected from the group consisting of collagen type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, and type XXVII. The human recombinant collagen can be collagen of one type free of any other type, or can be a mixture of collagen types. Suitably, the human recombinant collagen comprises collagens selected from the group consisting of collagen type I, collagen type III, and mixtures thereof. The term "bovine recombinant collagen" refers to collagen manufactured by culturing a non-human organism or mammalian or non-mammalian cells to express at least one bovine gene encoding a collagen. The bovine recombinant collagen may be selected from the group consisting of collagen type I, type II, type III, and type IV. The bovine recombinant collagen can be collagen of one type free of any other type, or can be a mixture of collagen types. Suitably, the bovine recombinant collagen comprises collagens selected from the group consisting of collagen type I, collagen type III, and mixtures thereof.

[0019] As understood by one of ordinary skill in the art,“molecular weight” (also known as “relative molar mass”) is a dimensionless quantity but is converted to molar mass by multiplying by 1 gram/mole - for example, collagen with a weight-average molecular weight of 5,000 has a weight- average molar mass of 5,000 g/mol.

[0020] As used herein, the term“biofilm” refers to an association of microorganisms, e.g. , single or multiple species, that can be encased or embedded in a matrix material, which may be self-produced by resident microorganisms. The biofdm may be present or adhere to living and/or non-living surfaces, e.g., tissue, a wound, medical implants, such as but not limited to orthopedic implants, dental implants, catheters, stents and so on. Exemplary microorganisms include, but are not limited to bacteria, e.g., Gram-negative bacteria, such as Pseudomonas aeruginosa, Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus mutans, and fungi, such as yeasts, e.g., Candida albicans. The term“matrix material” is intended to encompass extracellular polymeric substances. Exemplary matrix materials include, but are not limited to polysaccharides, glycoproteins and/or nucleic acids. The term“biofilm” is further intended to include biological fdms that develop and persist at interfaces in aqueous environments. The language“biofdm development” or“biofdm formation” is intended to include the formation, growth, and modification of the bacterial colonies contained with biofdm structures, as well as the synthesis and maintenance of the exopolysaccharide of the biofdm structures. “Reducing” or“disrupting” a biofdm includes reducing the number of total viable microorganisms making up at least part of the biofdm, for example, as measured by total viable counts (TVC) of microorganisms (e.g., bacteria, yeast). [0021] As used herein, the“administration” of a wound dressing composition to a subject includes any route of introducing or delivering to a subject a diagnostic wound dressing composition to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, topical administration. Administration includes self-administration and the administration by another.

[0022] As used herein, the term“effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g. , an amount which results in the decrease in a wound described herein or one or more signs or symptoms associated with a wound described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the wound and on the characteristics of the individual. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more wounds.

[0023] As used herein, the terms“individual”,“patient”, or“subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0024] “Treating” or“treatment” as used herein covers the treatment of a wound described herein, in a subject, such as a human, and includes: (i) inhibiting a wound, i.e., arresting its development; (ii) relieving a wound, i.e., causing regression of the wound; (iii) slowing progression of the wound; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the wound. In some embodiments, treatment means that the symptoms associated with the wound are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0025] It is also to be appreciated that the various modes of treatment of wounds as described herein are intended to mean“substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic wound and/or can include one or more administrations for the treatment of an acute wound.

The Wound Dressing Composition of the Present Technology

[0026] A wound can become infected by microbes. An infected wound is a wound in which bacteria or other microorganisms have colonized, causing a deterioration and delay in the healing of the wound. [0027] A biofilm is an association of microorganisms (e.g., single or multiple species) that can adhere to a surface forming three-dimensional microbial communities, which can have coordinated multi-cellular behavior. Typically, biofilms can produce extracellular polysaccharides thereby forming an extracellular matrix in which the bacteria are embedded. The ability of bacteria to form these complex biofilms can impede a host’s defense mechanisms against pathogens. As such, biofilms often display a heightened tolerance to antimicrobial treatment.

[0028] The present disclosure is directed to wound dressing compositions that include citric acid, sodium bicarbonate, or both, which may prevent, reduce, inhibit, or disrupt biofilm levels in a wound upon application, and over time. The wound dressing compositions of the present technology advantageously exhibit improved stability by sealing a citric acid-containing layer (or a sodium bicarbonate -containing layer, or a layer containing both) within other layers. Without being bound by theory, it is believed the improved stability is due, at least in part, to inhibiting the ability of the citric acid-containing layer (or a sodium bicarbonate-containing layer, or a layer containing both) from drawing in moisture from the air. The multi-layered construction of the wound compositions of the present technology may further be configured to provide a staged release of wound modulating components.

[0029] Thus, in an aspect, a wound dressing composition is provided that includes a first layer, a second layer, and a third layer. The first layer and third layer include a biopolymer. The second layer includes about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent that includes citric acid, sodium bicarbonate, or a combination thereof, and optionally a biopolymer (e.g., a mixture of collagen and oxidized regenerated cellulose (ORC)). In the wound dressing composition, the first layer and the third layer do not include citric acid (or do not include sodium bicarbonate, when sodium bicarbonate is present in the second layer), and the first layer and the third layer encapsulate the second layer.

[0030] In any embodiment herein, the first layer includes a biopolymer and a surface including a peripheral zone facing the third layer (“the first layer periphery”); the second layer includes a mixture of a collagen, an ORC, and about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent that includes citric acid, sodium bicarbonate, or a combination thereof; and the third layer includes a biopolymer and a surface that includes a peripheral zone facing the first layer (“the third layer periphery”); where the first layer and the third layer do not include citric acid (or do not include sodium bicarbonate, when sodium bicarbonate is present in the second layer), and the first layer periphery and the third layer periphery are adjoined to form a seal to provide the wound dressing composition - thus, the second layer is sealed within the wound composition. [0031] A“peripheral zone” of a surface is an area of extending inward from an external boundary (e.g., an outer edge) of the respective layer. The peripheral zone of any embodiment of either or both the first layer and the third layer may be an area extending inward from the external boundary by an average of about 0.1 cm to about 2.0 cm. Such an average may be determined by measuring the distance from the external boundary toward to center of the respective layer for at least 4 points on the external boundary that are equally distributed around the entire external boundary. Thus, the peripheral zone may extend inward an average of about 0.1 cm, about 0.2 cm, about 0.3 cm, about 0.4 cm, about 0.5 cm, about 0.6 cm, about 0.7 cm, about 0.8 cm, about 0.9 cm, about 1 cm, about 1.1 cm, about 1.2 cm, about 1.3 cm, about 1.4 cm, about 1.5 cm, about 1.6 cm, about 1.7 cm, about 1.8 cm, about 1.9 cm, about 2.0 cm, or any range including and/or in between any two of these values.

[0032] FIG. 1 provides a non-limiting representative illustration of an embodiment of each layer of the wound dressing composition 100 prior to adjoining the first layer 120 to the third layer 110 via the first layer periphery 150 and third layer periphery 140 and encapsulating the second layer 130.

[0033] FIG. 2 provides a non-limiting representative illustration of an embodiment of each layer of the wound dressing composition 200 after adjoining the first layer 220 to the third layer 210 via a seal 240 formed by the first layer periphery and third layer periphery such that the second layer 230 is sealed within the wound dressing composition 200.

The First Layer

[0034] In any embodiment disclosed herein, the first layer may include a wound-facing side and an environmental -facing side. The wound-facing side of the first layer may be configured to be in contact with a wound when in use. The first layer may be about 0.01 cm to about 0.5 cm thick; thus, the thickness of the first layer may be about 0.01 cm, about 0.02 cm, about 0.03 cm, about 0.04 cm, about 0.05 cm, about 0.06 cm, about 0.07 cm, about 0.08 cm, about 0.09 cm, about 0.10 cm, about 0.15 cm, about 0.20 cm, about 0.25 cm, about 0.30 cm, about 0.35 cm, about 0.40 cm, about 0.45 cm, about 0.50 cm, or any range including and/or in between any two of these values.

[0035] The biopolymer of first layer may be a bio-resorbable biopolymer such as collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, or a combination of any two or more thereof. The oxidized cellulose may be an oxidized regenerated cellulose (ORC) of any embodiment disclosed herein for the second layer. The first layer may include about 50 wt.% to about 100 wt.% of the biopolymer (based on the total weight of the first layer).

[0036] The collagen of the first layer may be a mammalian collagen. Additionally or alternatively, in some embodiments, the collagen of the first layer may comprise human collagen type I and human collagen type III. Additionally or alternatively, in some embodiments, the collagen of the first layer may comprise bovine collagen type I and bovine collagen type III.

[0037] In any embodiment disclosed herein, mammalian recombinant collagen of the first layer may be provided by any suitable method known in the art. Additionally or alternatively, in some embodiments, human recombinant collagen of the first layer may be provided by any suitable method known in the art. For example, the step of providing human recombinant collagen may comprise following the protocol described in U.S. Pat. No. 5,962,648, the entire content of which is incorporated herein by reference. Further recombinant processes are set forth in U.S. Pat. No.

5,593,859 and W02004/078120 which are also incorporated herein by reference. Additionally or alternatively, in some embodiments, collagen will be recombinantly manufactured by culturing a cell which has been transfected with at least one gene encoding a polypeptide comprising collagen and genes encoding oxidized cellulose and subunits of the post-translational enzyme prolyl 4-hydroxylase, and purifying the resultant collagen monomer therefrom. The human recombinant collagen solution may be subsequently subjected to polymerization or cross-linking conditions to produce an insoluble fibrous collagen.

[0038] In any embodiment disclosed herein, the first layer may include about 30 wt.% to 100 wt.% collagen where the collagen has a weight-average molecular weight of about 5,000 to about 100,000. Thus, the amount of collagen in the first layer may be about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, 100 wt.%, or any range including and/or in between any two of these values. Thus, the collagen in the first layer may be a weight-average molecular weight of about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 12,000, about 14,000, about 16,000, about 18,000, about 20,000, about 22,000, about 24,000, about 26,000, about 28,000, about 30,000, about 32,000, about 34,000, about 36,000, about 38,000, about 40,000, about 45,000, about 50,000, about 55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about 90,000, about 95,000, about 100,000, or any range including and/or in between any two of these values.

[0039] In any embodiment disclosed herein, the collagen of the first layer may comprise a weight ratio of human collagen type I to human collagen type III of about 100:0, about 90: 10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, about 10:90, about 0: 100, or any range including and/or in between any two of these values. Additionally or alternatively, in some embodiments, the ratio by weight of human collagen type I to human collagen type III is greater than about 50:50, or greater than about 70:30. Additionally or alternatively, in some embodiments, the collagen of the first layer may comprise a weight ratio of type I bovine collagen to type III bovine collagen of about 85: 15.

[0040] In any embodiment disclosed herein, oxidized regenerated cellulose (ORC) may be produced by the oxidation of cellulose, for example with dinitrogen tetroxide and/or as described in U.S. Pat. No. 3,122,479 (incorporated herein by reference). Without wishing to be bound by theory, it is believed that this process may convert primary alcohol groups on the saccharide residues of the cellulose to carboxylic acid groups, for example, forming uronic acid residues within the cellulose chain. The oxidation may not proceed with complete selectivity, and as a result hydroxyl groups on carbons 2 and 3 of the saccharide residue may be converted to the keto form. These ketone units may introduce an alkali labile link, which at pH 7 or higher initiates the decomposition of the polymer via formation of a lactone and sugar ring cleavage. As a result, oxidized regenerated cellulose is biodegradable and bioresorbable under physiological conditions. ORC is available with a variety of degrees of oxidation and hence rates of degradation. The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1000 pm, or from about 250 pm to about 450 pm.

[0041] In any embodiment disclosed herein, the first layer may include from about 30 wt.% to 100 wt.% of ORC, where the ORC may have a weight-average molecular weight of about 50,000 to about 1,000,000. Thus, ORC may be included in the first layer in an amount of about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, 100 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the ORC may include a weight-average molecular weight of about 50,000, about 55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about 90,000, about 95,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000, about 400,000, about 410,000, about 420,000, about 430,000, about 440,000, about 450,000, about 460,000, about 470,000, about 480,000, about 490,000, about 500,000, about 510,000, about 520,000, about 530,000, about 540,000, about 550,000, about 560,000, about 570,000, about 580,000, about 590,000, about 600,000, about 610,000, about 620,000, about 630,000, about 640,000, about 650,000, about 660,000, about 670,000, about 680,000, about 690,000, about 700,000, about 710,000, about 720,000, about 730,000, about 740,000, about 750,000, about 760,000, about 770,000, about 780,000, about 790,000, about 800,000, about 810,000, about 820,000, about 830,000, about 840,000, about 850,000, about 860,000, about 870,000, about 880,000, about 890,000, about 900,000, about 910,000, about 920,000, about 930,000, about 940,000, about 950,000, about 960,000, about 970,000, about 980,000, about 990,000, about 1,000,000, or any range including and/or in between any two of the preceding values.

[0042] The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1,000 pm. In any embodiment herein, the ORC may include fiber lengths of about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 pm, about 44 pm, about 46 pm, about 48 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 280 pm, about 300 pm, about 320 pm, about 340 pm, about 360 pm, about 380 pm, about 400 pm, about 420 pm, about 440 pm, about 460 pm, about 480 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1,000 pm, or any range including and/or in between any two of the preceding values.

[0043] In any embodiment disclosed herein, the first layer may include a weight ratio of collagen to ORC of about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:34, about 63:33, about 62:38, about 61 :39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51 :49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41 :59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31 :69, about 30:70, or any range including and/or in between any two of these values. Additionally or alternatively, in some embodiments, the weight ratio of the collagen to ORC may be about 55:45.

[0044] In any embodiment disclosed herein, the first layer may include at least one plasticizer. The at least one plasticizer may be included in the amount of about 0.001 wt.% to about 30 wt.% within the first layer; thus, the first layer may include plasticizers in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.5 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, about 12 wt.%, about 13 wt.%, about 14 wt.%, about 15 wt.%, about 16 wt.%, about 17 wt.%, about 18 wt.%, about 19 wt.%, about 20 wt.%, about 21 wt.%, about 22 wt.%, about 23 wt.%, about 24 wt.%, about 25 wt.%, about 26 wt.%, about 27 wt.%, about 28 wt.%, about 29 wt.%, about 30 wt.%, or any range including and/or in between any two of these values. For example, in any embodiment herein the first layer may include plasticizers in an amount form about 0.01 wt.% to about 5 wt.%. Exemplary plasticizers include, but are not limited to, an acetylated monoglyceride, an alkyl citrate, methyl ricinoleate, glycerol, or a combination of any two or more thereof. Examples of alkyl citrates include, but are not limited to, triethyl citrate, acetyl triethyl citrate, tributyl citrate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, acetyl trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, or a combination of any two or more thereof.

[0045] In any embodiment disclosed herein, the first layer may include about 0.001 wt.% to about 20 wt.% of a second antimicrobial agent. Thus, the second antimicrobial agent may be included in the first layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the second antimicrobial agent may be an ionic silver, silver acetate, a polyhexanide, chlorhexidine, iodine, or a combination of any two or more thereof.

[0046] In any embodiment disclosed herein, the first layer may comprise about 0.001 wt.% to about 20 wt.% of an antioxidant. Thus, the amount of antioxidant in the first layer may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary antioxidants include, but are not limited to, anthocyanins, astaxanthin, bilirubin, canthaxanthin, capsaicin, curcumin, coenzyme Q10, eugenol, flavanol, flavonolignans, flavanone, flavone, flavonol, iodide, isoflavone phytoestrogen, lutein, lycopene, manganese, melatonin, N- acetylcysteine, oxalic acid, phenolic acid, phytic acid, /Z-a-lipoic acid, stilbenoid, tocopherol, tocotrienol, vitamin A, vitamin C, vitamin E, xanthones, zeaxanthin, a-carotene, b-carotene, as well as a combination of any two or more thereof.

[0047] Examples of anthocyanins include, but are not limited to, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and mixtures of any two or more thereof.

[0048] Examples of flavanols include, but are not limited to, catechin, epicatechin, theaflavin, thearubigins, gallocatechin, epigallocatechin, a gallate ester of any thereof, and mixtures of any two or more thereof.

[0049] Examples of flavanones include, but are not limited to, eriodictyol, hesperetin, naringenin, and mixtures of any two or more thereof.

[0050] Examples of flavones include, but are not limited to, apigenin, luteolin, tangeritin, and mixtures of any two or more thereof.

[0051] Examples of flavonols include, but are not limited to, isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, and mixtures of any two or more thereof.

[0052] Examples of isoflavone phytoestrogens include, but are not limited to, daidzein, genistein, glycitein, and mixtures of any two or more thereof.

[0053] Examples of phenolic acids include, but are not limited to, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins, rosmarinic acid, salicylic acid, or any ester thereof, and mixtures of any two or more thereof.

[0054] Examples of stillbenoids include, but are not limited to, resveratrol, pterostilbene, and a mixture thereof. [0055] In any embodiment disclosed herein, the first layer may include about 0.001 wt.% to about 20 wt.% of at least one signaling protein. Thus, the at least one signaling protein may be included in the first layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary signaling proteins include, but are not limited to, an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a transforming growth factor beta (TGF ), or a combination of any two or more thereof.

The Second Layer

[0056] As provided previously, the second layer may include a biopolymer. For example, in any embodiment herein, the second layer may include a mixture of a collagen, an oxidized regenerated cellulose (ORC), and about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent that includes citric acid, sodium bicarbonate, or both. Thus, the amount of citric acid in the second layer (by total weight of the second layer) may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, or any range including and/or in between any two of these values, so long as the total amount of citric acid and sodium bicarbonate (when present) in the second layer is no more than about 10 wt.%.

[0057] Similarly, the amount of sodium bicarbonate in the second layer (by total weight of the second layer) may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, or any range including and/or in between any two of these values, so long as the total amount of sodium bicarbonate and citric acid (when present) in the second layer is no more than about 10 wt.% In any embodiment disclosed herein, the second layer may include a wound-facing side and an

environmental-facing side. The second layer may be about 0.01 cm to about 0.3 cm thick; thus, the thickness of the first layer may be about 0.01 cm, about 0.02 cm, about 0.03 cm, about 0.04 cm, about 0.05 cm, about 0.06 cm, about 0.07 cm, about 0.08 cm, about 0.09 cm, about 0.10 cm, about 0.15 cm, about 0.20 cm, about 0.25 cm, about 0.30 cm, or any range including and/or in between any two of these values.

[0058] In any embodiment herein, the second layer may include a biopolymer as described herein in any embodiment. The biopolymer of the second layer, in any embodiment herein, may be a biopolymer such as a bioresorbable polymer. Suitable biopolymers may include, but are not limited to, collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, or a combination of any two or more thereof. The oxidized cellulose may be an oxidized regenerated cellulose (ORC) of any embodiment disclosed herein for the second layer. In any embodiment herein, second layer may include a mixture of collagen and ORC. For example, the second layer may include a weight ratio of collagen to ORC of about 70:30 to about 30:70.

[0059] The collagen of the second layer may be a mammalian collagen. Additionally or alternatively, in some embodiments, the collagen of the second layer may comprise human collagen type I and human collagen type III. Additionally or alternatively, in some embodiments, the collagen of the second layer may comprise bovine collagen type I and bovine collagen type III.

[0060] In any embodiment disclosed herein, mammalian recombinant collagen of the second layer may be provided by any suitable method known in the art. Additionally or alternatively, in some embodiments, human recombinant collagen of the second layer may be provided by any suitable method known in the art. For example, the step of providing human recombinant collagen may comprise following the protocol described in U.S. Pat. No. 5,962,648, the entire content of which is incorporated herein by reference. Further recombinant processes are set forth in U.S. Pat. No.

5,593,859 and W02004/078120 which are also incorporated herein by reference. Additionally or alternatively, in some embodiments, collagen will be recombinantly manufactured by culturing a cell which has been transfected with at least one gene encoding a polypeptide comprising collagen and genes encoding oxidized cellulose and subunits of the post-translational enzyme prolyl 4-hydroxylase, and purifying the resultant collagen monomer therefrom. The human recombinant collagen solution may be subsequently subjected to polymerization or cross-linking conditions to produce an insoluble fibrous collagen. [0061] In any embodiment disclosed herein, the second layer may include about 30 wt.% to about 99.99 wt.% collagen where the collagen has a weight-average molecular weight of about 5,000 to about 100,000. Thus, the amount of collagen in the second layer may be about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, about 99.99 wt.%, or any range including and/or in between any two of these values. Thus, the collagen in the second layer may be a weight-average molecular weight of about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 12,000, about 14,000, about 16,000, about 18,000, about 20,000, about 22,000, about 24,000, about 26,000, about 28,000, about 30,000, about 32,000, about 34,000, about 36,000, about 38,000, about 40,000, about 45,000, about 50,000, about 55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about 90,000, about 95,000, about 100,000, or any range including and/or in between any two of these values.

[0062] In any embodiment disclosed herein, the collagen of the second layer may comprise a weight ratio of human collagen type I to human collagen type III of about 100:0, about 90: 10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, about 10:90, about 0: 100, or any range including and/or in between any two of these values. Additionally or alternatively, in some embodiments, the ratio by weight of human collagen type I to human collagen type III is greater than about 50:50, or greater than about 70:30. Additionally or alternatively, in some embodiments, the collagen of the second layer may comprise a weight ratio of type I bovine collagen to type III bovine collagen of about 85: 15.

[0063] In any embodiment disclosed herein, oxidized regenerated cellulose (ORC) may be produced by the oxidation of cellulose, for example with dinitrogen tetroxide and/or as described in U.S. Pat. No. 3,122,479 (incorporated herein by reference). Without wishing to be bound by theory, it is believed that this process may convert primary alcohol groups on the saccharide residues of the cellulose to carboxylic acid groups, for example, forming uronic acid residues within the cellulose chain. The oxidation may not proceed with complete selectivity, and as a result hydroxyl groups on carbons 2 and 3 of the saccharide residue may be converted to the keto form. These ketone units may introduce an alkali labile link, which at pH 7 or higher initiates the decomposition of the polymer via formation of a lactone and sugar ring cleavage. As a result, oxidized regenerated cellulose is biodegradable and bioresorbable under physiological conditions. ORC is available with a variety of degrees of oxidation and hence rates of degradation. The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1000 pm, or from about 250 pm to about 450 pm.

[0064] In any embodiment disclosed herein, the second layer may include from about 30 wt.% to about 99.99 wt.% of ORC, where the ORC may have a weight-average molecular weight of about 50,000 to about 1,000,000. Thus, ORC may be included in the second layer in an amount of about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, about 99.99 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the ORC may include a weight-average molecular weight of about 50,000, about 55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about 90,000, about 95,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000, about 400,000, about 410,000, about 420,000, about 430,000, about 440,000, about 450,000, about 460,000, about 470,000, about 480,000, about 490,000, about 500,000, about 510,000, about 520,000, about 530,000, about 540,000, about 550,000, about 560,000, about 570,000, about 580,000, about 590,000, about 600,000, about 610,000, about 620,000, about 630,000, about 640,000, about 650,000, about 660,000, about 670,000, about 680,000, about 690,000, about 700,000, about 710,000, about 720,000, about 730,000, about 740,000, about 750,000, about 760,000, about 770,000, about 780,000, about 790,000, about 800,000, about 810,000, about 820,000, about 830,000, about 840,000, about 850,000, about 860,000, about 870,000, about 880,000, about 890,000, about 900,000, about 910,000, about 920,000, about 930,000, about 940,000, about 950,000, about 960,000, about 970,000, about 980,000, about 990,000, about 1,000,000, or any range including and/or in between any two of the preceding values.

[0065] The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1,000 pm. In any embodiment herein, the ORC may include fiber lengths of about 5 mih, about 6 mih, about 7 mih, about 8 mih, about 9 mih, about 10 mih, about 11 mih, about 12 mih, about 13 mih, about 14 mih, about 15 mih, about 16 mih, about 17 mih, about 18 mm, about 19 mih, about 20 mm, about 22 mih, about 24 mih, about 26 mm, about 28 mih, about 30 mm, about 32 mih, about 34 mih, about 36 mm, about 38 mih, about 40 mm, about 42 mih, about 44 mih, about 46 mm, about 48 mih, about 50 mm, about 55 mih, about 60 mih, about 65 mm, about 70 mih, about 75 mm, about 80 mih, about 85 mih, about 90 mm, about 95 mih, about 100 mm, about 110 mih, about 120 mih, about 130 mm, about 140 mih, about 150 mm, about 160 mih, about 170 mih, about 180 mm, about 190 mih, about 200 mm, about 220 mih, about 230 mih, about 240 mm, about 250 mih, about 260 mm, about 280 mih, about 300 mih, about 320 mm, about 340 mih, about 360 mm, about 380 mih, about 400 mih, about 420 mm, about 440 mih, about 460 mm, about 480 mih, about 500 mih, about 550 mm, about 600 mih, about 650 mm, about 700 mih, about 750 mih, about 800 mm, about 850 mih, about 900 mm, about 950 mih, about 1,000 mih, or any range including and/or in between any two of the preceding values.

[0066] In any embodiment disclosed herein, the second layer may include a weight ratio of collagen to ORC of about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:34, about 63:33, about 62:38, about 61 :39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51 :49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41 :59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31 :69, about 30:70, or any range including and/or in between any two of these values. Additionally or alternatively, in some embodiments, the weight ratio of the collagen to ORC may be about 55:45.

[0067] In any embodiment disclosed herein, the second layer may include about 0.001 wt.% to about 20 wt.% of a second antimicrobial agent. Thus, the second antimicrobial agent may be included in the second layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the second antimicrobial agent may be an ionic silver, polyhexanide, chlorhexidine, iodine, or a combination of any two or more thereof. [0068] In any embodiment disclosed herein, the second layer may comprise about 0.001 wt.% to about 20 wt.% of an antioxidant. Thus, the amount of antioxidant in the second layer may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary antioxidants include, but are not limited to, anthocyanins, astaxanthin, bilirubin, canthaxanthin, capsaicin, curcumin, coenzyme Q10, eugenol, flavanol, flavonolignans, flavanone, flavone, flavonol, iodide, isoflavone phytoestrogen, lutein, lycopene, manganese, melatonin, N-acetylcysteine, oxalic acid, phenolic acid, phytic acid, R-a- lipoic acid, stilbenoid, tocopherol, tocotrienol, vitamin A, vitamin C, vitamin E, xanthones, zeaxanthin, a-carotene, b-carotene, as well as a combination of any two or more thereof.

[0069] Examples of anthocyanins include, but are not limited to, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and mixtures of any two or more thereof.

[0070] Examples of flavanols include, but are not limited to, catechin, epicatechin, theaflavin, thearubigins, gallocatechin, epigallocatechin, or any gallate ester thereof, and mixtures of any two or more thereof.

[0071] Examples of flavanones include, but are not limited to, eriodictyol, hesperetin, naringenin, and mixtures of any two or more thereof.

[0072] Examples of flavones include, but are not limited to, apigenin, luteolin, tangeritin, and mixtures of any two or more thereof.

[0073] Examples of flavonols include, but are not limited to, isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, and mixtures of any two or more thereof.

[0074] Examples of isoflavone phytoestrogens include, but are not limited to, daidzein, genistein, glycitein, and mixtures of any two or more thereof.

[0075] Examples of phenolic acids include, but are not limited to, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins, rosmarinic acid, salicylic acid, or any ester thereof, and mixtures of any two or more thereof. [0076] Examples of stillbenoids include, but are not limited to, resveratrol, pterostilbene, and mixtures thereof.

[0077] In any embodiment disclosed herein, the second layer may include about 0.001 wt.% to about 20 wt.% of at least one signaling protein. Thus, the at least one signaling protein may be included in the first layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary signaling proteins include, but are not limited to, an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a platelet- derived growth factor (PDGF), a transforming growth factor beta (TGF ), or a combination of any two or more thereof.

The Third Layer

[0078] As provided previously, the third layer includes a biopolymer and optionally a silver compound. In any embodiment disclosed herein, the third layer may include a wound-facing side and an environmental-facing side. The first layer may be about 0.01 cm to about 0.5 cm thick; thus, the thickness of the first layer may be about 0.01 cm, about 0.02 cm, about 0.03 cm, about 0.04 cm, about 0.05 cm, about 0.06 cm, about 0.07 cm, about 0.08 cm, about 0.09 cm, about 0.10 cm, about 0.15 cm, about 0.20 cm, about 0.25 cm, about 0.30 cm, about 0.35 cm, about 0.40 cm, about 0.45 cm, about 0.50 cm, or any range including and/or in between any two of these values.

[0079] The biopolymer of third layer may be a bio-resorbable polymer such as collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, or a combination of any two or more thereof. The oxidized cellulose may be an oxidized regenerated cellulose (ORC) of any embodiment disclosed herein for the second layer. The third layer may include about 50 wt.% to about 100 wt.% of the biopolymer.

[0080] The collagen of the third layer may be a mammalian collagen. Additionally or alternatively, in some embodiments, the collagen of the third layer may comprise human collagen type I and human collagen type III. Additionally or alternatively, in some embodiments, the collagen of the third layer may comprise bovine collagen type I and bovine collagen type III. [0081] In any embodiment disclosed herein, mammalian recombinant collagen of the third layer may be provided by any suitable method known in the art. Additionally or alternatively, in some embodiments, human recombinant collagen of the third layer may be provided by any suitable method known in the art. For example, the step of providing human recombinant collagen may comprise following the protocol described in U.S. Pat. No. 5,962,648, the entire content of which is incorporated herein by reference. Further recombinant processes are set forth in U.S. Pat. No.

[0082] In any embodiment disclosed herein, the third layer may include about 30 wt.% to about 100 wt.% collagen where the collagen has a weight-average molecular weight of about 5,000 to about 100,000. Thus, the amount of collagen in the third layer may be about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, 100 wt.%, or any range including and/or in between any two of these values. Thus, the collagen in the third layer may be a weight-average molecular weight of about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 12,000, about 14,000, about 16,000, about 18,000, about 20,000, about 22,000, about 24,000, about 26,000, about 28,000, about 30,000, about

32,000, about 34,000, about 36,000, about 38,000, about 40,000, about 45,000, about 50,000, about

55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about

90,000, about 95,000, about 100,000, or any range including and/or in between any two of these values.

[0083] In any embodiment disclosed herein, the collagen of the third layer may comprise a weight ratio of human collagen type I to human collagen type III of about 100:0, about 90: 10, about 80:20, about 70:30, about 60:40, about 50:50, about 40:60, about 30:70, about 20:80, about 10:90, about 0: 100, or any range including and/or in between any two of these values. Additionally or

alternatively, in some embodiments, the ratio by weight of human collagen type I to human collagen type III is greater than about 50:50, or greater than about 70:30. Additionally or alternatively, in some embodiments, the collagen of the third layer may comprise a weight ratio of type I bovine collagen to type III bovine collagen of about 85: 15.

[0084] In any embodiment disclosed herein, oxidized regenerated cellulose (ORC) may be produced by the oxidation of cellulose, for example with dinitrogen tetroxide and/or as described in U.S. Pat. No. 3,122,479 (incorporated herein by reference). Without wishing to be bound by theory, it is believed that this process may convert primary alcohol groups on the saccharide residues of the cellulose to carboxylic acid groups, for example, forming uronic acid residues within the cellulose chain. The oxidation may not proceed with complete selectivity, and as a result hydroxyl groups on carbons 2 and 3 of the saccharide residue may be converted to the keto form. These ketone units may introduce an alkali labile link, which at pH 7 or higher initiates the decomposition of the polymer via formation of a lactone and sugar ring cleavage. As a result, oxidized regenerated cellulose is biodegradable and bioresorbable under physiological conditions. ORC is available with a variety of degrees of oxidation and hence rates of degradation. The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1000 pm, or from about 250 pm to about 450 pm.

[0085] In any embodiment disclosed herein, the third layer may include from about 30 wt.% to about 100 wt.% of ORC, where the ORC may have a weight-average molecular weight of about 50,000 to about 1,000,000. Thus, ORC may be included in the third layer in an amount of about 30 wt.%, about 32 wt.%, about 34 wt.%, about 36 wt.%, about 38 wt.%, about 40 wt.%, about 42 wt.%, about 44 wt.%, about 46 wt.%, about 48 wt.%, about 50 wt.%, about 52 wt.%, about 54 wt.%, about 56 wt.%, about 58 wt.%, about 60 wt.%, about 62 wt.%, about 64 wt.%, about 66 wt.%, about 68 wt.%, about 70 wt.%, about 72 wt.%, about 74 wt.%, about 76 wt.%, about 78 wt.%, about 80 wt.%, about 82 wt.%, about 84 wt.%, about 86 wt.%, about 88 wt.%, about 90 wt.%, about 92 wt.%, about 94 wt.%, about 96 wt.%, about 98 wt.%, 100 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the ORC may include a weight-average molecular weight of about 50,000, about 55,000, about 60,000, about 65,000, about 70,000, about 75,000, about 80,000, about 85,000, about 90,000, about 95,000, about 100,000, about 110,000, about 120,000, about 130,000, about 140,000, about 150,000, about 160,000, about 170,000, about 180,000, about 190,000, about 200,000, about 210,000, about 220,000, about 230,000, about 240,000, about 250,000, about 260,000, about 270,000, about 280,000, about 290,000, about 300,000, about 310,000, about 320,000, about 330,000, about 340,000, about 350,000, about 360,000, about 370,000, about 380,000, about 390,000, about 400,000, about 410,000, about 420,000, about 430,000, about 440,000, about 450,000, about 460,000, about 470,000, about 480,000, about 490,000, about 500,000, about 510,000, about 520,000, about 530,000, about 540,000, about 550,000, about 560,000, about 570,000, about 580,000, about 590,000, about 600,000, about 610,000, about 620,000, about 630,000, about 640,000, about 650,000, about 660,000, about 670,000, about 680,000, about 690,000, about 700,000, about 710,000, about 720,000, about 730,000, about 740,000, about 750,000, about 760,000, about 770,000, about 780,000, about 790,000, about 800,000, about 810,000, about 820,000, about 830,000, about 840,000, about 850,000, about 860,000, about 870,000, about 880,000, about 890,000, about 900,000, about 910,000, about 920,000, about 930,000, about 940,000, about 950,000, about 960,000, about 970,000, about 980,000, about 990,000, about 1,000,000, or any range including and/or in between any two of the preceding values.

[0086] The ORC may include particles, fibers, or both; in any embodiment disclosed herein, the ORC may be in the form of particles, such as fiber particles or powder particles. In embodiments that include ORC fibers, the ORC fibers may have a volume fraction such that at least 80% of the fibers have lengths in the range from about 5 pm to about 1,000 pm. In any embodiment herein, the ORC may include fiber lengths of about 5 pm, about 6 pm, about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 22 pm, about 24 pm, about 26 pm, about 28 pm, about 30 pm, about 32 pm, about 34 pm, about 36 pm, about 38 pm, about 40 pm, about 42 pm, about 44 pm, about 46 pm, about 48 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 280 pm, about 300 pm, about 320 pm, about 340 pm, about 360 pm, about 380 pm, about 400 pm, about 420 pm, about 440 pm, about 460 pm, about 480 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1,000 pm, or any range including and/or in between any two of the preceding values.

[0087] In any embodiment disclosed herein, the third layer may include a weight ratio of collagen to ORC of about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:34, about 63:33, about 62:38, about 61 :39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51 :49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41 :59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31 :69, about 30:70, or any range including and/or in between any two of these values. Additionally or alternatively, in some embodiments, the weight ratio of the collagen to ORC may be about 55:45. [0088] In any embodiment disclosed herein, the third layer may include a silver compound. The silver compound (e.g., an ionic silver, silver acetate, silver citrate, or a combination of any two or more thereof) may be included in the amount of about 0.001 wt.% to about 20 wt.% within the third layer. Thus, total amount of silver compounds included in the third layer may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values.

[0089] In any embodiment disclosed herein, the third layer may include about 0.001 wt.% to about 20 wt.% of a second antimicrobial agent. Thus, the second antimicrobial agent may be included in the third layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. In any embodiment herein, the second antimicrobial agent may be a polyhexanide, chlorhexidine, iodine, or a combination of any two or more thereof.

[0090] In any embodiment disclosed herein, the third layer may include about 0.001 wt.% to about 20 wt.% of at least one signaling protein. Thus, the at least one signaling protein may be included in the third layer in an amount of about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary signaling proteins include, but are not limited to, EGF, FGF, PDGF, TGF , or a combination of any two or more thereof. [0091] In any embodiment disclosed herein, the third layer may comprise about 0.001 wt.% to about 20 wt.% of an antioxidant. Thus, the amount of antioxidant in the third layer may be about 0.001 wt.%, about 0.002 wt.%, about 0.003 wt.%, about 0.004 wt.%, about 0.005 wt.%, about 0.006 wt.%, about 0.007 wt.%, about 0.008 wt.%, about 0.009 wt.%, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, about 0.05 wt.%, about 0.06 wt.%, about 0.07 wt.%, about 0.08 wt.%, about 0.09 wt.%, about 0.1 wt.%, about 0.2 wt.%, about 0.3 wt.%, about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, about 10 wt.%, about 15 wt.%, about 20 wt.%, or any range including and/or in between any two of these values. Exemplary antioxidants include, but are not limited to, anthocyanins, astaxanthin, bilirubin, canthaxanthin, capsaicin, curcumin, coenzyme Q10, eugenol, flavanol, flavonolignans, flavanone, flavone, flavonol, iodide, isoflavone phytoestrogen, lutein, lycopene, manganese, melatonin, N- acetylcysteine, oxalic acid, phenolic acid, phytic acid, /Z-a-lipoic acid, stilbenoid, tocopherol, tocotrienol, vitamin A, vitamin C, vitamin E, xanthones, zeaxanthin, a-carotene, b-carotene, as well as a combination of any two or more thereof.

[0092] Examples of anthocyanins include, but are not limited to cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and mixtures of any two or more thereof.

[0093] Examples of flavanols include, but are not limited to catechin, epicatechin, theaflavin, thearubigins, gallocatechin, epigallocatechin, or any gallate ester thereof, and mixtures of any two or more thereof.

[0094] Examples of flavanones include, but are not limited to eriodictyol, hesperetin, naringenin, and mixtures of any two or more thereof.

[0095] Examples of flavones include, but are not limited to apigenin, luteolin, tangeritin, and mixtures of any two or more thereof.

[0096] Examples of flavonols include, but are not limited to isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, and mixtures of any two or more thereof.

[0097] Examples of isoflavone phytoestrogens include, but are not limited to daidzein, genistein, glycitein, and mixtures of any two or more thereof.

[0098] Examples of phenolic acids include, but are not limited to chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins, rosmarinic acid, salicylic acid, or any ester thereof, and mixtures of any two or more thereof. [0099] Examples of stillbenoids includes, but is not limited to resveratrol, pterostilbene, and mixtures of any two or more thereof.

The Wound Dressing

[0100] As provided previously, the present disclosure provides a wound dressing composition comprising a first layer, a second layer, and a third layer, wherein each of the first layer, the second layer, and the third layer comprise a wound-facing side and an environmental -facing side.

[0101] In any embodiment disclosed herein, the wound-facing side of the third layer is in contact and/or adjoined with the environmental-facing side of the second layer, and the wound-facing side of the second layer is in contact and/or adjoined with the environmental-facing side of the first layer. In any embodiment herein, while the second layer may be in contact with the environment-facing side of the first layer and the wound-facing side of the third layer as described herein, the second layer may not be bound to the first layer and/or the third layer. As used herein, the term“bound” refers to the first layer, second layer, and/or third layer being attached via any connective means, such as via an adhesive layer or formation of a continuous polymer matrix.

[0102] Additionally or alternatively, in some embodiments, the first layer and the third layer may form a continuous outer layer encapsulating the second layer within the wound dressing composition. For example, the outer layer may be a continuous matrix of biopolymer as described herein for the first layer and/or third layer in any embodiment. FIG. 3 shows a cross-sectional view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology, where the first layer 320 and third layer 310 form a continuous outer layer encapsulating the second layer 330.

[0103] Additionally or alternatively, in some embodiments, the first layer, the second layer, and the third layer may form a continuous polymer matrix, where the first antimicrobial agent that includes about 0.001 wt.% to about 10 wt.% of citric acid, sodium bicarbonate, or a combination thereof is concentrated within the second layer of the wound dressing. FIG. 4 shows a cross-sectional view of a diagrammatic representation of an embodiment of a wound dressing composition of the present technology, where the first layer 420, third layer 410, and second layer 430 form a continuous biopolymer matrix where the first antimicrobial agent is entirely concentrated within the second layer 430.

[0104] In any embodiment herein, the first layer and/or third layer me be a film having a suitable thickness from about 100 pm to about 2000 pm; thus, in any embodiment herein, the film may have a thickness of about 100 pm, about 200 pm, about 300 pm, about 400 pm, about 500 pm, about 600 pm, about 700 pm, about 800 pm, about 900 pm, about 1000 pm, about 1100 pm, about 1200 pm, about 1300 mih, about 1400 mih, about 1500 mih, about 1600 mih, about 1700 mih, about 1800 mih, about 1900 mih, about 2000 mih, or any range including and/or in between any two of the preceding values.

[0105] In any embodiment disclosed herein, the wound dressing composition of the present disclosure may be sterile and packaged in a microorganism-impermeable container.

[0106] In any embodiment disclosed herein, the wound dressing composition of the present technology may be capable of preventing, reducing, inhibiting, or disrupting biofdm formation in a wound. Reducing a biofilm includes reducing the number of total viable microorganisms making up at least part of the biofdm, for example, as measured by total viable counts (TVC) of microorganisms (e.g., bacteria, yeast). The biofdm may comprise bacteria including, but not limited to, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus mutans. The biofdm may also include fungi including but not limited to yeasts, such as Candida albicans. Additionally or alternatively, in some embodiments, the wound dressing composition of the present technology may be capable of preventing, reducing, inhibiting, or disrupting a biofdm in a wound by > about 10% to about 100%, after 24 hours in vitro exposure. Additionally or alternatively, in some embodiments, the wound dressing composition of the present technology may be capable of preventing, reducing, inhibiting, or disrupting a biofdm in a wound by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, about 100%, or any range including and/or in between any two of these values.

[0107] The therapeutic efficacy of the wound dressing composition of the present technology can be assayed using any method known to those in the art. An exemplary method to test the therapeutic efficacy of the wound dressing composition of the present technology is the colony drip flow reactor (C-DFR) assay ( see Lipp, C., el al., J. Wound Care, 19:220-226(2010), which is incorporated herein by reference).

Optional Additional Components or Lavers

[0108] In any embodiment herein, the wound dressing composition may include one or more additional layers. The one or more additional layers may be configured to perform any of a variety of functions including, for example, adherence of the dressing to the wound or to surrounding tissues, increasing structural rigidity of the dressing, protection of the dressing from moisture or other materials in the external environment, protection of a wound surface, delivery of one or more active materials or other materials to the wound surface, or a combination of two or more thereof. In any embodiment herein, the additional layers may be conformable to a wound surface and/or to the surrounding tissues. For example, in any embodiment herein, the one or more additional layers may be configured to be capable of bending such that the wound-facing surfaces of the dressing are in substantial contact with the wound and/or the surrounding tissues.

[0109] In any embodiment herein, the first layer, the second layer, and/or third layer may be in contact with a surface of the one or more additional layers. Additionally or alternatively, in some embodiments, the first layer and/or third layer may be in contact with a surface of the one or more additional layers. In any embodiment herein, the one or more additional layers may include a polyurethane foam, an absorbent foam, a non-adherent layer, a protective layer, or a tissue contact layer.

[0110] In any embodiment herein, the one or more additional layers may be a cover. In any embodiment herein, the cover may be permeable to water vapor and impermeable or substantially impermeable to liquid. For example, in any embodiment herein, the cover is not permeable to liquid water or wound exudate. The cover may have a moisture vapor transmission rate (MVTR) of about 300 g/m²/24 hours to about 5000 g/m²/24 hours, such as from about 500 g/m²/24 hours to about 2000 g/m²/24 hours at 37.5°C at 50% relative humidity difference as described in ASTM E96-00; thus, the MVTR may be about 500 g/m²/24 hours, about 750 g/m²/24 hours, about 1000 g/m²/24 hours, about 1500 g/m²/24 hours, about 2000 g/m²/24 hours, about 2500 g/m²/24 hours, about 3000 g/m²/24 hours, about 3500 g/m²/24 hours, about 4000 g/m²/24 hours, about 4500 g/m²/24 hours, about 5000 g/m²/24 hours, or any range including and/or in between any two of the preceding values. In any embodiment herein, the cover may be impermeable or substantially impermeable to microorganisms.

[0111] The cover may be formed from polymers. Suitable polymers for forming the cover may include, but are not limited to, polyurethanes, poly alkoxyalkyl acrylates, methacrylates, or a combination of any two or more thereof. For example, in any embodiment herein, the cover may include a continuous layer of a high-density blocked polyurethane foam that is predominantly closed cell. Suitable cover materials (e.g., polymers) are disclosed in U.S. Patent 3,645,835 issued February 29, 1972, incorporated by reference herein in its entirety. In any embodiment herein, the cover material may be the polyurethane film commercially available as Estane® 5714F (sold by The Lubrizol Corporation).

[0112] The cover may have a thickness in the range from about 10 pm to about 1000 pm, such as from about 100 pm to about 500 pm; thus, the cover may have a thickness of about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, 100 pm, about 150 pm, about 200 pm, about 250 pm, about 300 pm, about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1000 pm, or any range including and/or in between any two of the preceding values. The surfaces of the cover may have a size and configuration such that the cover extends beyond the dressing and defines a marginal region extending from about 0.5 mm to about 60 mm, such as from about 1 mm to about 50 mm; thus, in any embodiment herein, the marginal region may extend from about 0.5 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, about 50 mm, about 55 mm, about 60 mm, or any range including and/or in between any two or more of the preceding values. In any embodiment herein, the cover may extend beyond one or more edges of the dressing, including the first layer and/or the third layer. In any embodiment herein, the cover may be configured such that a portion of the marginal area around the dressing of the cover may be coated with an adhesive layer, such that when applied to a wound tissue, the marginal area may be used to adhere the dressing to tissues surrounding the wound.

[0113] Suitable adhesives may include, but are not limited to, pressure sensitive adhesives. For example, in any embodiment herein, the pressure sensitive adhesive may include acrylate ester copolymers, polyvinyl ethyl ether, polyurethane, or a combination of any two or more thereof.

Suitable pressure sensitive adhesives include, but are not limited to, those pressure sensitive adhesives disclosed in U.S. Patent 3,645,835, issued February 29, 1972, incorporated herein by reference in its entirety. The adhesive layer may have a basis weight of about 20 g/m² to about 250 g/m².

[0114] In any embodiment herein, the one or more additional layers may include fluid pathways interconnected so as to improve distribution or collection of fluids. For example, in any embodiment herein, the secondary layer may be a porous foam material having a plurality of interconnected cells, pores, edges, walls, or a combination of two or more thereof to form interconnected fluid pathways (e.g., channels). Suitable porous foam materials may include, but are not limited to, cellular foam, open-cell foam, reticulated foam, porous tissue collections, other porous materials (e.g., gauze or felted mat), or a combination of two or more thereof. In any embodiment herein, the one or more additional layers may be a foam having pore sizes in of about 400 microns, about 420 microns, about 440 microns, about 460 microns, about 480 microns, about 500 microns, about 520 microns, about 540 microns, about 560 microns, about 580 microns, about 600 microns, or any range including and/or in between any two of the preceding values. Thus, in any embodiment herein, the one or more additional layers may be an open-cell, reticulated polyurethane foam.

[0115] The one or more additional layers may be characterized as exhibiting absorbency. For example, in any embodiment herein, the one or more additional layers may exhibit an absorbency of at least about 3 g saline/g, at least about 4 g saline/g, at least about 5 g saline/g, at least about 6 g saline/g, at least about 7 g saline/g, at least about 8 g saline/g, at least about 9 g saline/g, at least about 10 g saline/g, at least about 11 g saline/g, at least about 12 g saline/g, at least about 13 g saline/g, at least about 14 g saline/g, at least about 15 g saline/g, at least about 16 g saline/g, at least about 17 g saline/g, at least about 18 g saline/g, at least about 19 g saline/g, at least about 20 g saline/g, or any range including and/or in between any two of the preceding values. The one or more additional layers may be hydrophilic and configured to absorb (e.g., wick) fluid away from the dressing. For example, in any embodiment herein, the wicking properties of the one or more additional layers may draw fluid away from the dressing by capillary flow or other wicking mechanisms. Suitable hydrophilic foams include, but are not limited to, a polyvinyl alcohol, open-cell foam, hydrophilic foams made from polyether, hydrophobic foams that have been treated or coated to provide hydrophilicity, or a combination of any two or more thereof.

Methods of Making the Wound Dressing of the Present Technology

[0116] Based on the description of the wound dressing of the present technology, a person of ordinary skill in the art will understand how to generate compositions of the present technology.

[0117] Simply by way of illustration, an exemplary wound dressing composition according to some embodiments of the present technology - where, e.g. , the first and third layers include the same components and the second layer also include such components with the further inclusion of citric acid and/or sodium bicarbonate - may be generated as described in this paragraph. The first layer may be prepared by initially generating a slurry, where the slurry contains 0.5 - 2% w/v collagen swelled in 0.05 M acetic acid, 0.4-1% w/v ORC, and glycerol (present at 50pl-2ml per 100ml of slurry). Collagen present in the slurry at 0.5 - 2% w/v, ORC present at 0.4-1% w/v and glycerol present at 50pl-2ml per 100ml of slurry. These materials are combined using a mixing device and subsequently degassed in a vacuum to remove trapped air. The mixture is poured into a tray (about 0.3 lg/cm²) and then dehydrated (e.g., at a temperature of about 37°C for 24 hours). The second layer may be generated in a similar fashion with the exception that citric acid and/or sodium bicarbonate is included in the slurry at a concentration of about 100 mM to about 400mM and the slurry can be a non-aqueous slurry (e.g., an alcohol or organic solvent can be used as the liquid phase). The second layer may be cut to two-dimensional size (length and width) that is smaller than or equal to the two- dimensional size of the first layer. The first layer may be adhered to the second layer by placing together the surface of each layer that was in contact with the bottom of the respective tray used in dehydration of each layer. The adhered first and second layers may then be placed in a same sized tray with the first layer contacting the bottom of the tray. The third layer may then be made in a similar fashion as the first layer and adhered to the second layer or the same amount of slurry as used for the first layer poured over the adhered first and second layers, followed by dehydration (e.g., at a temperature of about 37°C for 24 hours). The procedure may be performed or modified to result in adhering the first layer to the second layer and the second layer to the third layer and/or sealing the first and third layers to each other (e.g., at a peripheral zone), with the second layer adhered or not adhered to either one or both of the first and third layers. Thus, the procedure as described herein provides a wound dressing composition with multiple layers.

Negative-Pressure Therapy

[0118] The dressing of any embodiment described herein may be employed in therapy in which a wound is treated with reduced pressure. Treatment of tissue with reduced pressure may be commonly referred to as“negative-pressure therapy,” but is also known by other names, including“negative- pressure wound therapy,”“reduced-pressure therapy,”“vacuum therapy,”“vacuum-assisted closure,” and“topical negative-pressure,” for example. Negative-pressure therapy may provide a number of benefits, including migration of epithelial and subcutaneous tissues, improved blood flow, and/or micro-deformation of tissue at a wound site. Together, these benefits may increase development of granulation tissue and reduce healing times.

[0119] Generally, the system may be configured to provide negative-pressure to a wound in accordance with this specification. In any embodiment herein, the system may generally include a negative-pressure supply, and may include or be configured to be coupled to a distribution component. In general, a distribution component may refer to any complementary or ancillary component configured to be fluidly coupled to a negative -pressure supply in a fluid path between a negative- pressure supply and a wound.

[0120] In any embodiment herein, the dressing may be configured to distribute negative pressure. The dressing may comprise or be configured as a manifold. A“manifold” in this context generally includes any composition or structure providing a plurality of pathways configured to collect or distribute fluid across a wound under pressure. For example, a manifold may be configured to receive negative pressure from the negative-pressure source and to distribute negative pressure through multiple apertures (e.g., pores), which may have the effect of collecting fluid and drawing the fluid toward the negative-pressure source. Additionally or alternatively, the fluid path(s) may be reversed or a secondary fluid path may be provided to facilitate movement of fluid across a wound. Additionally or alternatively, the fluid pathways of a manifold may be interconnected to improve distribution or collection of fluids. Additionally or alternatively, a manifold may be a porous material having a plurality of interconnected cells or pores. For example, in any embodiment herein, open-cell foams such as reticulated foams may generally include pores, edges, and/or walls that may form interconnected fluid pathways (such as channels).

[0121] The fluid mechanics associated with using a negative-pressure source to reduce pressure in another component or location, such as within a sealed therapeutic environment, can be mathematically complex. However, the basic principles of fluid mechanics applicable to negative-pressure therapy are generally well-known to those skilled in the art. The process of reducing pressure may be described generally and illustratively herein as“delivering,”“distributing,” or“generating” negative pressure, for example.

[0122] In general, a fluid, such as wound fluid (for example, wound exudates and other fluids), flows toward lower pressure along a fluid path. Thus, the term“downstream” typically implies something in a fluid path relatively closer to a source of negative pressure or further away from a source of positive pressure. Conversely, the term“upstream” implies something relatively further away from a source of negative pressure or closer to a source of positive pressure. This orientation is generally presumed for purposes of describing various features and components herein. However, the fluid path may also be reversed in some applications (such as by substituting a positive-pressure source for a negative-pressure source) and this descriptive convention should not be construed as a limiting convention.

[0123] “Negative pressure” may generally refer to a pressure less than a local ambient pressure, such as the ambient pressure in a local environment external to a sealed therapeutic environment provided by the dressing. In many cases, the local ambient pressure may also be the atmospheric pressure proximate to or about a wound. Alternatively or additionally, the pressure may be less than a hydrostatic pressure associated with the tissue at the wound. While the amount and nature of negative pressure applied to a wound may vary according to therapeutic requirements, the pressure is generally a low vacuum, also commonly referred to as a rough vacuum, between -5 mm Hg (-667 Pa) and -500 mm Hg (-66.7 kPa), gauge pressure. Common therapeutic ranges are between -50 mm Hg (-6.7 kPa) and -300 mm Hg (-39.9 kPa), gauge pressure.

[0124] Additionally or alternatively, in any embodiment herein, a negative-pressure supply may be a reservoir of air at a negative pressure, or may be a manual or electrically-powered device that can reduce the pressure in a sealed volume, such as a vacuum pump, a suction pump, a wall suction port available at many healthcare facilities, or a micro-pump, for example. A negative-pressure supply may be housed within or used in conjunction with other components, such as sensors, processing units, alarm indicators, memory, databases, software, display devices, or user interfaces that further facilitate therapy. A negative-pressure source may be combined with a controller and other components into a therapy unit. A negative-pressure supply may also have one or more supply ports configured to facilitate coupling and de-coupling of the negative-pressure supply to one or more distribution components.

[0125] In any embodiment herein, components may be fluidly coupled to each other to provide a path for transferring fluids (i.e.. liquid and/or gas) between the components. For example, components may be fluidly coupled through a fluid conductor, such as a tube. As used herein, the term“fluid conductor” may include a tube, pipe, hose, conduit, or other structure with one or more lumina or open passages adapted to convey a fluid between two ends thereof. Typically, a fluid conductor may be an elongated, cylindrical structure with some flexibility, but the geometry and rigidity may vary. Additionally or alternatively, in any embodiment herein, the negative-pressure source may be operatively coupled to the dressing via a dressing interface.

Treatment Methods of the Present Technology

[0126] In an aspect, methods for treating a wound in a subject in need thereof are provided, wherein the method includes administering to the wound a wound dressing composition of any embodiment disclosed herein. The wound may be an acute wound or a chronic wound. Exemplary chronic wounds include, but are not limited to, infectious wounds, venous ulcers, decubitis ulcers, and/or diabetic ulcers; exemplary acute wounds include, but are not limited to, surgical wounds, trauma wounds, bum wounds, and/or donor sites. In any embodiment disclosed herein, the wound dressing composition may protect the wound from infection. The infection may be a bacterial infection or a fungal infection. The infection may include infection with a biofdm. In any embodiment disclosed herein, the bacterial infection may be caused by gram-negative or gram-positive bacteria.

[0127] Examples of gram-positive bacteria include, but are not limited to, Actinomyces sp., Arcanohacterium sp., Bacillus sp., Bavariicoccus sp., Brachyhacterium sp., Clostridium sp.,

Cnuihacter sp., Corynehacterium sp., Enterococcus sp., Desulfitohacterium sp., Fervidohacterium sp., Georgenia sp., Janihacter sp., Lactohacillales sp.. Microbispora sp., Nocardia sp., Pasteuria sp., Pilihacter sp., Propionihacterium sp., Rathayibacter sp., Rhodococcus sp., Rosehuria sp., Rothia sp., Sarcina sp., Solihacillus sp., Sporosarcina sp., Staphylococcus sp., Streptococcus sp.,

Syntrophomonas sp., or Tepidihacter sp.

[0128] Examples of gram-negative bacteria include, but are not limited to, Acetobacter sp., Acidaminococcus sp., Acinetobacter s ., Agrobacterium sp., Akkermansia sp ., Anaerobiospirillum sp., Anaerolinea sp., Arcobacter sp., Armatimonas sp., Azotobacter sp., Bacteroides sp., Bacteroidetes sp., Bartonella sp., Bdellovibriosp., Brachyspira sp., Bradyrhizobium sp., Caldilinea sp., Cardiobacterium sp., Christensenella sp., Chthonomonas sp., Coxiella sp., Cyanobacteria sp., Cytophaga sp.,

Dehalogenimonas sp., Desulfurobacterium sp., Devosia sp., Dialister sp., Dictyoglomus sp.,

Dinoroseobacter sp., Enterobacter sp., Escherichia sp., Fimbriimonas sp., Flavobacterium sp., Francisella sp., Fusobacterium sp., Gluconacetobacter sp., Haemophilus sp., Helicobacter sp., Kingella sp., Klebsiella sp., Kluyvera sp., Kozakia sp., Legionella sp. Leptonema sp. Leptotrichia sp., Levilinea sp. Luteimonas sp. Megamonas sp., Megasphaera sp., Meiothermus sp ., Methylobacterium sp., Moraxella sp., Morganella sp., Mycoplasma sp., Neisseria sp., Nitrosomonas sp., Pectinatus sp., Pedobacter sp., Pelosinus sp., Propionispora sp., Proteus sp., Pseudomonas sp., Pseudoxanthomonas sp., Rickettsia sp., Salinibacter sp., Salmonella sp., Samsonia sp., Serratia sp., Shigella sp., Shimwellia sp., Sphingomonas sp., Stenotrophomonas sp., Thorselliaceae sp., Vampirococcus sp., Verminephrobacter sp., Vibrio sp., Victivallis sp., Vitreoscilla sp., Wolbachia sp.

[0129] In any embodiment disclosed herein, the infection may be caused by Aspergillus sp., Aureobasidium sp., Candida sp., Cladosporium sp., Curvularia sp., Engodontium sp., Epicoccum sp., Gibberella sp., Hypocreales sp., Leptosphaerulina sp., Malessezia sp., Penicillium sp.,

Rhodosporidium sp., Trichosporon sp., Trichtophyton sp., and Ulocladium sp.

[0130] In any embodiment disclosed herein, the wound may include a biofdm, and the wound dressing composition of the present technology prevents, reduces, inhibits, or disrupts the biofdm.

[0131] Additionally or alternatively, in some embodiments, the wound dressing composition is administered directly to the wound. Any method known to those in the art for administering a wound dressing composition to an acute or a chronic wound disclosed herein may be employed. Suitable methods include in vitro or in vivo methods. In vivo methods typically include the administration of one or more wound dressing compositions to a subject in need thereof, suitably a human. When used in vivo for therapy, the one or more wound dressing compositions described herein are administered to the subject in effective amounts (i.e.. amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the state of the wound of the subject, and the characteristics of the particular wound dressing composition used.

[0132] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more wound dressing compositions useful in the methods may be administered to a subject in need thereof by any number of well-known methods for administering wound dressing compositions.

[0133] In any embodiment disclosed herein, the wound dressing compositions may be administered daily for 1 hour or more, for 2 hours or more, for 3 hours or more, for 4 hours or more, for 5 hours or more, for 6 hours or more, for 12 hours or more. In any embodiment disclosed herein, the wound dressing compositions may be administered one, two, three, four, or five times per day. In any embodiment disclosed herein, the wound dressing compositions may be administered daily for one, two, three, four or five weeks. In any embodiment disclosed herein, the wound dressing compositions may be administered daily for less than 6 weeks. In any embodiment disclosed herein, the wound dressing compositions may be administered daily for 6 weeks or more. In any embodiment disclosed herein, the wound dressing compositions may be administered daily for 12 weeks or more. In any embodiment disclosed herein, the wound dressing compositions may be administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In any embodiment disclosed herein, the wound dressing compositions may be administered weekly, bi weekly, tri-weekly, or monthly. In any embodiment disclosed herein, the wound dressing compositions may be administered for a period of one, two, three, four, or five weeks. In any embodiment disclosed herein, the wound dressing compositions may be administered for six weeks or more. In any embodiment disclosed herein, the wound dressing compositions may be administered for twelve weeks or more. In any embodiment disclosed herein, the wound dressing compositions may be administered for a period of less than one year. In any embodiment disclosed herein, the wound dressing compositions may be administered for a period of more than one year. In any embodiment disclosed herein, the wound dressing compositions may be administered for a chronic wound as appropriate.

[0134] In any embodiment herein, the process may further include employing the wound dressing in the context of a negative-pressure therapy, where the negative-pressure therapy may include positioning the wound dressing proximate to the wound. For example, the various components of the dressing may be positioned with respect to the wound sequentially or, alternatively, may be positioned with respect to each other and then positioned with respect to the wound. The negative-pressure therapy may further comprise sealing the wound dressing to tissue surrounding the wound to form a sealed space. For example, the wound dressing may be positioned proximate to the wound and sealed to an attachment surface near the wound, for example, to undamaged epidermis peripheral to a wound.

[0135] The negative-pressure therapy method in any embodiment herein may further include fluidly coupling a negative-pressure source to the sealed space and operating the negative-pressure source to generate a negative pressure in the sealed space. For example, the negative-pressure source may be coupled to the dressing such that the negative-pressure source may be used to reduce the pressure in the sealed space. For example, negative pressure applied across the wound, for example, via the dressing may be effective to induce macrostrain and microstrain at the wound, as well as remove exudates and other fluids from the wound.

[0136] The present technology also provides a method of preventing formation of a biofilm or reducing biofilm at a wound, where the method includes applying a wound dressing composition of any embodiment herein of the present technology adjacent to the wound.

Kits Comprising the Wound Dressing of the Present Technology

[0137] In a further related aspect, the present disclosure provides kits that include a wound dressing composition of any embodiment described herein and instructions for use. The kits of the present technology may also include methods for treating a wound in a subject in need thereof. The kit may optionally comprise components such as antiseptic wipes, ointment, adhesive tape, tweezers, scissors, etc. EXPERIMENTAL EXAMPLES

[0138] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1: Disrupting Biofilms in a Wound.

[0139] A wound dressing composition of any embodiment herein will be administered directly to a chronic wound, with the wound-facing side of the first layer contacting the wound. A first biofilm level will be determined using the colony drip flow reactor (C-DFR), described herein. A second biofilm level will be determined 72 hours after administering the wound dressing composition to the wound. It is anticipated that administration of the wound dressing composition to a chronic wound will result in the prevention, reduction, inhibition, or disruption of biofilm levels in the wound.

[0140] These results will demonstrate that the wound dressing compositions of the present technology are useful for preventing, reducing, inhibiting, or disrupting biofilm levels in a wound in a subject in need thereof.

EQUIVALENTS

[0141] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0142] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0143] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,”“greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0144] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Claims

1. A wound dressing composition comprising:

a first layer comprising a biopolymer and a surface comprising a peripheral zone facing a third layer (“the first layer periphery”);

a second layer comprising a mixture of a collagen, an oxidized regenerated cellulose (ORC), and about 0.001 wt.% to about 10 wt.% a first antimicrobial agent comprising citric acid and/or sodium bicarbonate; and

the third layer comprising a biopolymer and a surface comprising a peripheral zone facing the first layer (“the third layer periphery”); and

wherein

the first layer and the third layer do not comprise citric acid or sodium bicarbonate; and

the first layer periphery and the third layer periphery are adjoined to form a seal.

2. The wound dressing composition of claim 1, wherein the biopolymer of the first layer is selected from the group consisting of a collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, and a combination of any two or more thereof.

3. The wound dressing composition of claim 1 or claim 2, wherein the biopolymer of the first layer comprises about 50 wt.% to about 100 wt.% of the first layer.

4. The wound dressing composition of any one of claims 1-3, wherein a weight ratio of collagen to ORC in the second layer is about 70:30 to about 30:70.

5. The wound dressing composition of any one of claims 1-4, wherein the biopolymer of the third layer is selected from the group consisting of a collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, and a combination of any two or more thereof.

6. The wound dressing composition of any one of claims 1-5, wherein the biopolymer of the third layer comprises about 50 wt.% to about 100 wt.% of the third layer.

7. The wound dressing composition of any one of claims 1-6, wherein the wound dressing further comprises a silver compound.

8. The wound dressing of claim 7, wherein the silver compounds comprise an ionic silver, silver acetate, silver citrate, or a combination of any two or more thereof.

9. The wound dressing composition of any one of claims 1-8, wherein the first layer, second layer, and/or third layer further comprises at least one plasticizer.

10. The wound dressing composition of claim 9, wherein the first layer, second layer, and/or third layer comprises about 0.01 wt.% to about 30 wt.% of at least one plasticizer.

11. The wound dressing composition of claim 10, wherein the at least one plasticizer is selected from the group consisting of an acetylated monoglyceride, an alkyl citrate, methyl ricinoleate, glycerol, and a combination of any two or more thereof.

12. The wound dressing composition of claim 11, wherein the alkyl citrate is triethyl citrate, acetyl triethyl citrate, tributyl citrate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, acetyl trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, or a combination of any two or more thereof.

13. The wound dressing composition of any one of claims 1-12, wherein the first layer, the second layer, and/or the third layer further comprises about 0.001 wt.% to about 20 wt.% of a second antimicrobial agent.

14. The wound dressing composition of claim 13, wherein the second antimicrobial agent is selected from the group consisting of an ionic silver, polyhexanide, chlorhexidine, and iodine.

15. The wound dressing composition of any one of claims 1-14, wherein the first layer and/or third layer further comprises about 0.001 wt.% to about 20 wt.% of a signaling protein.

16. The wound dressing composition of claim 15, wherein the signaling protein is selected from the group consisting of an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a transforming growth factor beta (TGF ), and a combination of any two or more thereof.

17. The wound dressing composition of any one of claims 1-16, wherein the first layer, the second layer, and/or the third layer further comprises about 0.001 wt.% to about 5 wt.% of an antioxidant.

18. The wound dressing composition of claim 17, wherein the antioxidant is selected from the group consisting of anthocyanins, astaxanthin, bilirubin, canthaxanthin, capsaicin, curcumin, coenzyme Q10, eugenol, flavanol, flavonolignans, flavanone, flavone, flavonol, iodide, isoflavone phytoestrogen, lutein, lycopene, manganese, melatonin, N-acetylcysteine, oxalic acid, phenolic acid, phytic acid, /Z-a-lipoic acid, stilbenoid, tocopherol, tocotrienol, vitamin A, vitamin C, vitamin E, xanthones, zeaxanthin, a-carotene, b-carotene, and a combination of any two or more thereof.

19. The wound dressing composition of claim 18, wherein the anthocyanins are selected from the group consisting of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and a combination of any two or more thereof.

20. The wound dressing composition of claim 18, wherein the flavanol is catechin, epicatechin,

theaflavin, thearubigins, gallocatechin, epigallocatechin, or any gallate ester thereof, or a combination of any two or more thereof.

21. The wound dressing composition of claim 18, wherein the flavanone is eriodictyol, hesperetin, naringenin, or a combination of any two or more thereof.

22. The wound dressing composition of claim 18, wherein the flavone is apigenin, luteolin, tangeritin, or a combination of any two or more thereof.

23. The wound dressing composition of claim 18, wherein the flavonol is isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, or a combination of any two or more thereof.

24. The wound dressing composition of claim 18, wherein the isoflavone phytoestrogen is daidzein, genistein, glycitein, or a combination of any two or more thereof.

25. The wound dressing composition of claim 18, wherein the phenolic acid is chicoric acid,

chlorogenic acid, cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins, rosmarinic acid, salicylic acid, or an ester of any thereof, or a combination of any two or more thereof.

26. The wound dressing composition of claim 18, wherein the stilbenoid is resveratrol, pterostilbene, or a combination thereof.

27. The wound dressing composition of any one of claims 1-26, wherein the first layer and/or third layer are in the form of a film.

28. The wound dressing composition of claim 27, wherein the film has a thickness of about 100 pm to about 2000 pm.

29. The wound dressing composition of any one of claims 1-28, further comprising one or more

additional layers, wherein the first layer and/or third layer is in contact with a surface of the one or more additional layers.

30. The wound dressing composition of claim 29, wherein the one or more additional layer comprises a polyurethane foam, an absorbent foam, a non-adherent layer, a protective layer, or a tissue contact layer.

31. The wound dressing composition of claim 29 or claim 30, wherein the one or more additional layers is a cover layer.

32. The wound dressing composition of any one of claims 1-31, wherein the wound dressing

composition is capable of preventing, reducing, inhibiting or disrupting biofdm formation in a wound.

33. The wound dressing composition of any one of claims 1-32, wherein the wound dressing

composition is capable of reducing a biofdm by about 10% or greater after 24 hours in vitro exposure.

34. A method for treating a wound in a subject in need thereof, the method comprising administering to the wound a wound dressing composition of any one of claims 1-33.

35. The method of claim 34, wherein the wound dressing composition is administered directly to the wound.

36. The method of claim 34 or claim 35, wherein the wound dressing composition prevents, reduces, inhibits, or disrupts biofilm formation in a wound.

37. The method of any one of claims 34-36, further comprising providing negative pressure therapy to the wound.

38. A kit comprising

the wound dressing composition of any one of claims 1-33, and

instructions for use.

39. A wound dressing composition comprising:

a first layer;

a second layer comprising about 0.001 wt.% to about 10 wt.% of a first antimicrobial agent comprising citric acid and/or sodium bicarbonate; and

the third layer; and

wherein

the first layer and/or third layer comprise a mixture of collagen and oxidized

regenerated cellulose (ORC);

the first layer and the third layer encapsulate the second layer.

40. The wound dressing composition of claim 39, wherein the first layer comprises a biopolymer selected from the group consisting of a collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin and a combination of any two or more thereof.

41. The wound dressing composition of claim 39 or claim 40, wherein the biopolymer of the first layer comprises about 50 wt.% to about 100 wt.% of the first layer.

42. The wound dressing composition of any one of claims 39-41, wherein the third layer comprises a biopolymer selected from the group consisting of a collagen, an oxidized cellulose, a

polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, and a combination of any two or more thereof.

43. The wound dressing composition of any one of claims 39-42, wherein the biopolymer of the third layer comprises about 50 wt.% to about 100 wt.% of the third layer.

44. The wound dressing composition of any one of claims 39-43, wherein the first layer and/or third layer comprises a weight ratio of collagen to ORC of about 70:30 to about 30:70.

45. The wound dressing composition of any one of claims 39-44, wherein the first layer and third layer together form a continuous outer layer encapsulating the second layer.

46. The wound dressing composition of any one of claims 39-45, wherein the second layer further comprises a biopolymer selected from the group consisting of a collagen, an oxidized cellulose, a polysaccharide, chitosan, gelatin, hyaluronic acid, elastin, fibronectin, and a combination of any two or more thereof.

47. The wound dressing composition of any one of claims 39-46, wherein the first layer comprises a surface comprising a peripheral zone facing the third layer (“the first layer periphery”) and the third layer comprises a surface comprising a peripheral zone facing the first layer (“the third layer periphery”).

48. The wound dressing composition of claim 47, wherein the first layer periphery and the third layer periphery are adjoined to form a seal.

49. The wound dressing composition of any one of claims 39-48, further comprising a silver

compound, wherein the silver compound comprises an ionic silver, silver acetate, silver citrate, or a combination of any two or more thereof.

50. The wound dressing composition of any one of claims 39-49, wherein the first layer and/or the third layer further comprises an at least one plasticizer.

51. The wound dressing composition of claim 50, wherein the first layer, second layer, and/or third layer comprises about 0.01 wt.% to about 30 wt.% of at least one plasticizer.

52. The wound dressing composition of claim 51, wherein the at least one plasticizer is selected from the group consisting of an acetylated monoglyceride, an alkyl citrate, methyl ricinoleate, glycerol, and a combination of any two or more thereof.

53. The wound dressing composition of claim 52, wherein the alkyl citrate is triethyl citrate, acetyl triethyl citrate, tributyl citrate, acetyl tributyl citrate, trioctyl citrate, acetyl trioctyl citrate, trihexyl citrate, acetyl trihexyl citrate, butyryl trihexyl citrate, trimethyl citrate, or a combination of any two or more thereof.

54. The wound dressing composition of any one of claims 39-53, wherein the first layer, the second layer, and/or the third layer further comprises about 0.001 wt.% to about 20 wt.% of a second antimicrobial agent.

55. The wound dressing composition of claim 54, wherein the second antimicrobial agent is selected from the group consisting of an ionic silver, polyhexanide, chlorhexidine, and iodine.

56. The wound dressing composition of any one of claims 39-55, wherein the first layer and/or the third layer further comprises about 0.001 wt.% to about 20 wt.% of a signaling protein.

57. The wound dressing composition of claim 56, wherein the signaling protein is selected from the group consisting of an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a transforming growth factor beta (TGF ), and a combination of any two or more thereof.

58. The wound dressing composition of any one of claims 39-57, wherein the first layer, the second layer, and/or the third layer further comprises about 0.001 wt.% to about 5 wt.% of an antioxidant.

59. The wound dressing composition of claim 58, wherein the antioxidant is selected from the group consisting of anthocyanins, astaxanthin, bilirubin, canthaxanthin, capsaicin, curcumin, coenzyme Q10, eugenol, flavanol, flavonolignans, flavanone, flavone, flavonol, iodide, isoflavone phytoestrogen, lutein, lycopene, manganese, melatonin, N-acetylcysteine, oxalic acid, phenolic acid, phytic acid, /Z-a-lipoic acid, stilbenoid, tocopherol, tocotrienol, vitamin A, vitamin C, vitamin E, xanthones, zeaxanthin, a-carotene, b-carotene, and a combination of any two or more thereof.

60. The wound dressing composition of claim 59, wherein the anthocyanins are selected from the group consisting of cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and a combination of any two or more thereof.

61. The wound dressing composition of claim 59, wherein the flavanol is catechin, epicatechin,

62. The wound dressing composition of claim 59, wherein the flavanone is eriodictyol, hesperetin, naringenin, or a combination of any two or more thereof.

63. The wound dressing composition of claim 59, wherein the flavone is apigenin, luteolin, tangeritin, or a combination of any two or more thereof.

64. The wound dressing composition of claim 59, wherein the flavonol is isorhamnetin, kaempferol, myricetin, proanthocyanidins, quercetin, rutin, or a combination of any two or more thereof.

65. The wound dressing composition of claim 59, wherein the isoflavone phytoestrogen is daidzein, genistein, glycitein, or a combination of any two or more thereof.

66. The wound dressing composition of claim 59, wherein the phenolic acid is chicoric acid,

67. The wound dressing composition of claim 59, wherein the stilbenoid is resveratrol, pterostilbene, or a combination thereof.

68. The wound dressing composition of any one of claims 39-67, wherein the first layer and/or third layer are in the form of a film.

69. The wound dressing composition of claim 68, wherein the film has a thickness of about 100 pm to about 2000 pm.

70. The wound dressing composition of any one of claims 39-69, further comprising one or more additional layers, wherein the first layer and/or third layer is in contact with a surface of the one or more additional layers.

71. The wound dressing composition of claim 70, wherein the one or more additional layer comprises a polyurethane foam, an absorbent foam, a non-adherent layer, a protective layer, or a tissue contact layer.

72. The wound dressing composition of claim 70 or claim 71, wherein the one or more additional layers is a cover layer.

73. The wound dressing composition of any one of claims 39-72, wherein the wound dressing

composition is capable of preventing, reducing, inhibiting or disrupting biofilm formation in a wound.

74. The wound dressing composition of any one of claims 39-73, wherein the wound dressing

75. A method for treating a wound in a subject in need thereof, the method comprising administering to the wound a wound dressing composition of any one of claims 39-74.

76. The method of claim 75, wherein the wound dressing composition is administered directly to the wound.

77. The method of claim 75 or claim 76, wherein the wound dressing composition prevents, reduces, inhibits, or disrupts biofilm formation in a wound.

78. The method of any one of claims 75-77, further comprising providing negative pressure to the wound.

79. A kit comprising

the wound dressing composition of any one of claims 39-74, and

instructions for use.