PRIORITY INFORMATION

[0001] The present application claims priority to U.S. Provisional Patent

Application No. 62/511,723 filed on May 26, 2017, which is incorporated by reference herein.

GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under NIDCR R01 - DEO 19355 awarded by NIH. The government has certain rights in the invention.

FIELD OF TECHNOLOGY

[0003] A multi-layered system is generally provided that is designed to reduce healing time of difficult to heal wounds as well as treat enterocutaneous and enteroatmospheric fistulas. Such a therapeutic device has broad application in the wound healing market.

BACKGROUND

[0004] Difficult-to-heal wounds are a tremendous health care burden, including chronic wounds, non-healing wounds, physiologically challenging wounds, etc. Such difficult-to-heal wounds are increasingly common and pose a major dilemma in the clinical setting, and may include diabetic wounds, pressure wounds, venous ulcers, and others. In the US, chronic wounds account for more than $25 billion annually in healthcare expenses and affect 6.5 million people. Health care expenditures for wound care are eclipsing other chronic medical conditions such as chronic obstructive pulmonary disease. For example, pressure ulcers are one of the most common types of difficult-to-heal wounds, and account for billions of dollars annually to the US health care system.

[0005] Wound healing is a highly orchestrated series of events with acute innate inflammation occurring soon after hemostasis. While this early inflammatory response serves to clear the injured area of bacteria, it also causes collateral damage to host tissues primarily through excessive neutrophil infiltration, release of hydrolysis enzymes, and reactive oxygen species. Paradoxically, over exuberant acute inflammation can cause collateral tissue necrosis that cannot be resolved due to poor vascular supply and impaired cell migration which interrupts normal progression of the wound healing cycle. Recent literature as well as our own published studies show that purinergic signaling (ATP signaling) plays a key role in neutrophil attraction and targeting.

[0006] The wound exudate is generated as part of the inflammatory response, and in a healthy wound is essential to the wound healing process. In chronic wounds, the exudate inhibits cell proliferation, damages healthy cells, and contains high levels of enzymes such as MPO and MMPs that have molecular weights of 15-50 KDa.

Traditional treatment strategies dictate the adequate management of the wound exudate, which contains a large amount of inflammatory signals to promote the normal wound healing cascade. In some cases, these methodologies are not sufficient.

[0007] Ubiquitous opportunistic microorganisms readily occupy the site of an open wound. The presence of these microorganisms exacerbates the already unresolved inflammatory response. Strategies that preemptively exclude these organisms would improve the ability to accelerate closure.

[0008] While numerous advanced wound dressing devices have been developed and are currently on the market, they don't address as a whole the multifactorial contributors that arrest the wound healing cascade in these difficult to heal wounds. Instead, the primary purpose of traditional wound dressings is to control wound effluent and prevent bacteria from lodging at the injury site. The utilization of current wound healing devices is labor intensive, cost aversive, induces discomfort, and fails to primarily address many of the factors which contribute to delays in wound healing. Specifically, difficult-to-heal wounds are impaired due to poor oxygenation, chronic inflammation, and many times infection resulting in degradation of growth factors, poor cell migration, and disruption of the wound healing cycle. Finally, one of the main limitations of conventional wound healing dressings is that they fail to optimize the wound healing environment and require frequent replacement, resulting in a painful, expensive, and labor-intensive process. [0009] As such, traditional wound healing devices fall short in addressing the key limitations of difficult to heal wound healing. For example, traditional wound healing devices fail to address chronic pressure ulcers since such wounds are a multifactorial problem that includes: inflammation, impaired angiogenesis, copious wound exudate, diminished cell migration, irregular moisture balance within the wound bed, and bacterial loads.

[0010] Thus, a need exists for improved wound healing devices and methods for the treatment of chronic wounds.

BRIEF DESCRIPTION

[0011] Aspects and advantages will be set forth in part in the following description, or may be obvious from the description, or may be learned through practice of the invention.

[0012] A wound dressing is generally provided. In one embodiment, the wound dressing includes a top layer comprising a fibrous sheet formed from a plurality of fibers. The plurality of fibers are comprised of reaction electrospun collagen fibers, regenerated cellulose fibers, hydrophobic polymeric fibers, or a mixture thereof. In particular embodiments, the top layer may comprise at least two fibrous sheets laminated together and/or comprise a coating of silver on its top side. For example, a coating of silver may define an exposed surface of the wound dressing such that the top layer has a contact angle with water that is about 90° or greater.

[0013] In particular embodiments, the plurality of fibers are comprised of polymeric fibers (e.g., polypropylene, polyethylene, polyester, polyurethane, polycarbonate, polytetrafluoroethylene, expanded-PTFE, polyethersulfone, polybutylene succinate, polydioxanone, polyglycolic acid, or a mixture thereof).

[0014] In particular embodiments, the wound dressing further includes a bottom layer comprising electrospun hydrophilic collagen fibers, wherein the top layer is on the bottom layer. For example, the bottom layer may further comprise antiinflammatory peptides on or within the electrospun hydrophilic collagen fibers. For instance, the bottom layer may include JM2, IL-IRa CYT-658, mefloquine, suramin, C34, CR2-Crry, Crry-Ig, anti-Co mAb, C3a receptor, C5a receptor antagonist, CR2- fH, anti-fB and CR2-CD59, or a mixture thereof. The bottom layer may be shaped to the depth of the wound.

[0015] In some embodiments, the top layer has a diameter that is greater than the bottom layer. For example, the top layer may define an overhang portion configured to adhere to skin surrounding the wound.

[0016] An inner layer may be, in one embodiment, positioned between the top layer and the bottom layer. For example, the inner layer may comprise synthetic granulation tissue, such as fibroblast, endothelial cells, parenchymal, immune cells, macrophages, smooth muscle cells, or mixtures thereof. Such an inner layer may have a thickness of about 0.5 mm to about 2 mm.

[0017] Methods of treating a wound are also generally provided. In one embodiment, the method may include applying the wound dressing onto a wound of a subjection with the top layer exposed (e.g., the silver layer, when present). As such, the bottom layer (when present) may be directly in contact with the exposed tissue within the wound.

[0018] Methods are also generally provided for forming a wound dressing. In one embodiment, the method includes forming a coating of silver nanoparticles on a first surface of a top layer; forming an inner layer on the second surface of the top layer; and applying a bottom layer onto the inner layer. In one embodiment, the top layer includes at least two fibrous sheets laminated together with one of at least one of the fibrous sheets comprising a plurality of fibers comprised of reaction electrospun collagen fibers, regenerated cellulose fibers, polymeric fibers, or a mixture thereof. The inner layer may include synthetic granulation tissue. The bottom layer may comprise electrospun hydrophilic collagen fibers.

[0019] These and other features, aspects and advantages will become better understood with reference to the following description and appended claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain certain principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] A full and enabling disclosure of the present invention, including the best mode thereof, directed to one of ordinary skill in the art, is set forth in the

specification, which makes reference to the accompanying figures.

[0021] FIG. 1 A is a diagram showing a cross-section of an exemplary wound dressing system that includes a silver coated top layer, an optional inner layer, and a bottom layer having optional active agents attached thereon (depicted as P).

[0022] FIG. IB is a diagram showing a cross-section of an exemplary wound dressing system that includes a silver coated top layer formed from multiple layers, an optional inner layer, and a bottom layer having optional active agents attached thereon (depicted as P).

[0023] FIG. 2A is a diagram showing a cross-section of an exemplary wound dressing system that includes a silver coated top layer formed from multiple layers.

[0024] FIG. 2B is a diagram showing a cross-section of an exemplary wound dressing system that includes a silver coated top layer formed from multiple layers and a bottom layer having optional active agents attached thereon (depicted as P).

[0025] FIG. 3 A shows scanning electron microscope (SEM) images of silver a woven polyester membrane sputter-coated with silver at 30mAmp@125s.

[0026] FIG. 3B shows SEM images of a hydrophilic 0.1 μπι PTFE membrane.

[0027] FIG. 4A shows an image taken for contact angle analysis of a silver coated woven polyester membrane sputter coated with the parameter 30mAmp@150s.

[0028] FIG. 4B shows an image taken for contact angle analysis of a silver coated woven polyester membrane sputter coated with the following parameter

30mAmp@125s.

[0029] FIG. 5A shows the antimicrobial properties of the silver coated woven polyester membrane sputter coated at 30mAmp@125s as demonstrated by a Kirby- Bauer test in a Methicillin-resistant Staphylococcus aureus (MRSA) bacterial colony.

[0030] FIG. 5B shows the antimicrobial properties of the silver coated woven polyester membrane sputter coated at 30mAmp@125s as demonstrated by a Kirby- Bauer test in a E. Coli bacterial colony.

[0031] FIG. 5C shows the antimicrobial properties of the silver coated woven polyester membrane sputter coated at 30mAmp@125s as demonstrated by a Kirby- Bauer test in a methicillin-susceptible Staphylococcus aureus iSSA) bacterial colony.

[0032] FIG. 6 shows a graph of the fluorescence intensity from the 96-well plate according to the Examples. Cells metabolize the blue non-fluorescent dye to red fluorescent resorufin. Therefore, an approximation of the amount of cells alive in each well can be extrapolated from the fluorescence intensity.

[0033] FIG. 7 shows an exemplary system for imaging a wound and producing an electrospun bottom layer that generally conforms dimensionally (e.g., in thickness and in width) with the wound.

[0034] FIG. 8A shows an early assembly of a wound dressing with multiple layers according to an example.

[0035] FIG. 8B shows a completed, trimmed assembly of a wound dressing with multiple layers according to an example.

[0036] FIG. 9 A shows TEM image of reaction electrospun collagen fibers and 9B show SEM images of reaction electrospun collagen fibers.

[0037] FIG. 9C shows an immunohistochemistry image of a reaction electrospun fibers seeded with endothelial cells and fibroblasts with nuclei (Hoescht), F actin (Phalloidin), and endothelial networks (indicated with CD31).

[0038] FIG. 10A, FIG. 10B, and FIG. IOC show the tissue engineered living pre- vascular network composed of 4: 1 fibroblasts to endothelial cells, SPEC.

[0039] FIG. 11 shows the moisture content of two exemplary wound dressing systems having differing collagen fibers.

[0040] FIG. 12 shows the bulk absorption of two exemplary wound dressing systems (uncoated vs. silver coated).

[0041] FIG. 13 shows various images of an exemplary wound dressing system.

DEFINITIONS

[0042] Chemical elements are discussed in the present disclosure using their common chemical abbreviation, such as commonly found on a periodic table of elements. For example, hydrogen is represented by its common chemical

abbreviation H; helium is represented by its common chemical abbreviation He; and so forth. [0043] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random and alternating copolymers; and terpolymers; and blends and modifications thereof.

Furthermore, unless otherwise specifically limited, the term "polymer" shall include all possible geometrical configurations of the material. These configurations include, but are not limited to isotactic, syndiotactic, and random symmetries.

[0044] As used herein, the prefix "nano" refers to the nanometer scale up to about 100 nm. For example, particles having an average diameter on the nanometer scale (e.g., from about 0.1 nm to about 100 nm) are referred to as "nanoparticles."

[0045] In the present disclosure, when a layer is being described as "on" or "over" another layer or substrate, it is to be understood that the layers can either be directly contacting each other or have another layer or feature between the layers, unless expressly stated to the contrary. Thus, these terms are simply describing the relative position of the layers to each other and do not necessarily mean "on top of since the relative position above or below depends upon the orientation of the device to the viewer.

DETAILED DESCRIPTION OF PARTICULAR EMBODFMENTS

[0046] Reference now will be made in detail to embodiments of the invention, one or more examples of which are illustrated in the drawings. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used with another embodiment to yield a still further

embodiment. Thus, it is intended that the present invention covers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0047] A wound dressing is generally provided, along with methods of its formation and use in treating a wound. Generally, the wound dressing is a multilayered system that constitutes a smart wound matrix. In certain embodiments, the wound dressing addresses the limitations of previous wound dressing devices by: (1) reducing the inflammatory response, (2) providing cues to induce angiogenesis and cellularization of the wound, (3) allowing for unidirectional wound exudate drainage, (4) regulates moisture balance within the wound bed, and/or (5) creates a semi-permanent biodegradable wound dressing. For example, the wound dressing may provide an improved wound healing environment by minimizing the bioburden, promoting cell migration, increasing neovascularization, and/or transporting wound edema away from injury while regulating the moisture balance in the wound bed.

[0048] In certain embodiments, the wound dressing also addresses the primary challenge facing tissue engineering, namely, development of constructs that promote neovascularization. In a wound healing environment, at least a portion of the wound dressing serves as scaffold material to provide the necessary stimulus to the surrounding tissue to promote perfusion of the construct to remove waste and C02 while providing oxygen and nutrients and augmenting cellular infiltration. In one embodiment, the scaffold material is a tissue engineered living layer created according to the recently developed scaffold-free technology by the present inventors' research group (referred to as the elf-organizing Prevascularized Endothelial- fibroblast Constructs (SPEC)). These constructs are generally engineered to contain a prevascular bed and supply reparative fibroblasts so as to provide cues to encourage migration and repopulation of the wound bed with viable host cells as well as to promote neovascularization. For example, a Reaction Electrospun Collagen (RESC) fiber layer may line the wound bed and serve a dual function: first providing a provisional matrix to host cell migration, and second to regulate moisture balance in the wound bed.

[0049] The wound dressing may also provide wound effluent control while simultaneously preventing bacterial over-growth to enhance healing. As such, the wound dressing may provide a wound healing environment using surface modified hydrophobic groups to transport wound edema away from the injury. In addition, the issue of dressing removal may be ameliorated by the wound dressing because it is composed of mostly bioabsorbable materials. The amphipathic nature of this layer creates a unidirectional flow of liquid out of the wound bed, thereby creating an appropriate environment that promotes regeneration, while simultaneously preventing bacteria from entering the wound. In addition, the hydrophobic outer silver coating kills invading pathogens and provides an added layer of protection against infection. [0050] In summary, the smart wound matrix is particularly useful for difficult to heal wounds and provides an approach to an extremely important clinical entity. The matrix may also prevent deleterious inflammation (e.g., may reduce the inflammatory response via purinergic signaling blockade to promote and accelerate the healing process), provides effluent control (e.g., may provide a unidirectional fluid flux barrier layer for wound fluid to egress) and regulates moisture levels within the wound bed (e.g., hydrophilic collagen fibers regulate moisture balance and promote exudate flow out of the wound), enhances neovascularization (e.g., may induce neovascularization), augments cellular migration, prevents infection (e.g., may reduce bioburden with silver coated surface or incorporation of antibiotic compounds), speeds epithelialization, and/or avoids dressing-removal altogether. The wound dressing may induce the regeneration cascade without the need to be removed by using biodegradable materials. This innovative combination of both new and proven technologies has the potential to significantly improve outcomes to a number of commonly encountered clinical wounds from tissue defects from necrotizing infection, burns, lacerations, pressure wounds, dermal ulcers, to enterocutaneous and enteroatmospheric fistulas.

[0051] I. Wound Dressing

[0052] FIG. 1A shows an exemplary wound dressing 10 as a multilayer system including: a top layer 12, an optional inner layer 14, and an optional bottom layer 16. In one particular embodiment, the wound dressing 10 includes three layers: a hydrophilic bottom layer 16, which in certain embodiments is coated with active materials (P) such as anti-inflammatory peptides and/or anti-biotic compounds, an inner layer 14 of synthetic granulation tissue, and a hydrophobic top layer 12 (i.e., an outer layer). Generally, the top layer 12 defines an external surface 13 of the dressing 10, while the bottom layer 16 forms a wound-facing surface 17. FIG. IB is similar to FIG. 1 A, with the top layer 12 including multiple layers, shown as an external outer layer 18 and an inner outer layer 20.

[0053] In certain embodiments, such as shown in FIG. 2A, the top layer 12 may be utilized alone without the inner layer 14 and the bottom layer 16. Additionally, as shown in FIG. 2B, the top layer 12 may be utilized with the bottom layer 16, without the inner layer 14. [0054] Each of these layers is discussed in greater detail below. It is noted that the optional inner layer and the bottom layer are generally bio-compatible and do not have to be removed from the wound after the healing process is complete.

[0055] A. Top Layer

[0056] The top layer 12 includes a porous fibrous sheet, and generally facilitates the passage of wound exudate through the membrane (i.e., away from the wound) while retaining larger proteins and cells within the wound bed. As such, the top layer provides wound effluent control to the wound dressing by controlling the movement of liquid out of the wound bed through capillary action. In particular embodiments, the top layer is configured to behave similarly to an ultrafiltration membrane with a 100 kDa molecular weight cut off. The top layer serves as a protective layer and may slough off as the wound heals.

[0057] In one embodiment, the top layer 12 is generally composed of a fibrous sheet formed from a plurality of fibers. The fibrous sheet may be a woven sheet or a nonwoven sheet of the fibers. The fibers may be composed of reaction electrospun collagen fibers, regenerated cellulose fibers, or polymeric fibers. For example, particularly suitable polymeric fibers may include polypropylene, polyethylene, polyester, polyurethane, polycarbonate, polytetrafluoroethylene (PTFE), expanded- PTFE (ePTFE), polyethersulfone, polybutylene succinate (PBS), polydioxanone (PDO, which may also be referred to as polydioxanone suture or PDS), polyglycolic acid (PGA), and mixtures thereof.

[0058] As shown in Fig. IB, two fibrous sheets may be utilized to form, together, the top layer 12, shown as the external outer layer 18 and an inner outer layer 20. Such a multilayer top layer 12 is particularly suitable for use with an inner layer 16 of reaction electrospun collagen fibers, regenerated cellulose fibers, hydrophilic polymeric fibers (e.g., polypropylene, polyethylene, polyester, polyurethane, polycarbonate, polytetrafluoroethylene (PTFE), expanded-PTFE (ePTFE), polyethersulfone, polybutylene succinate (PBS), polydioxanone (PDO, which may also be referred to as polydioxanone suture or PDS), polyglycolic acid (PGA)), or mixtures thereof and an outer layer of polymeric fibers (coated on the opposite side with Ag nanoparticles). [0059] The thickness (T _t ) of the top layer 12 is generally designed to be sufficient to protect the underlying wound (e.g., providing antimicrobial properties) while allowing for wound effluent control. For example, the top layer 12 may have a thickness (T _t ) that is about 20 μπι to about 10 mm, and in certain embodiments may have a thickness (T _t ) that is about 25 μπι to about 1 mm (e.g., about 25 μπι to about 100 μιη).

[0060] In particular embodiments, the fibrous sheet of the top layer 12 is silver (Ag) coated (shown as Ag nanoparticles 22) on its outer surface 13 to help with the antibacterial properties of the top layer 12 and also to control its efflux properties, such as by sputter coating methods. These hydrophobic silver nanoparticles 22 bonded to the top layer 12 of the matrix simultaneously create anti -bacterial surface that prevent infection and retards the development of bacterial laden biofilm of the injured tissue. The silver nanoparticles 22 also adds hydrophobicity to the outer surface of the underlying fibrous sheet.

[0061] The combination of the fibrous sheet coated on the outer surface 13 with the silver particles gives the membrane transport properties of a hydrophilic ultrafiltration side (facing the wound) and a hydrophobic antibacterial side (facing the air) to keep external fluids and microorganisms from transporting into the wound bed while providing wound effluent control. The amphipathic nature of this layer creates a unidirectional flow of liquid out of the wound bed, thereby creating an appropriate environment that promotes regeneration, while simultaneously preventing bacteria from entering the wound.

[0062] In one embodiment, the outer surface 13 of the top layer 12 (i.e., defined by the silver particles 22) has a hydrophobicity that influences the contact angle of the outer surface 13 of the top layer 12 so as to inhibit aqueous solutions from flowing into the wound. As such, the outer surface 13 of the top layer 12 provides

hydrophobicity to the wound dressing 10. In certain embodiments, the outer surface 13 of the top layer 12 has a contact angle with water that is about 90° or greater (e.g., about 100° to about 175°), such as shown in the exemplary dressings of FIGS. 4A and 4B. In particular embodiments, the contact angle of the outer surface 13 can be controlled through the concentration of the silver nanoparticles 22 on thereon. Even though certain materials of the top layer 12 may have hydrophilic properties, the top layer 12 may remain hydrophobic due to the materials or combination of materials therein, particularly with respect to the outer surface 13 coated with Ag nanoparticles 22.

[0063] As shown in FIGS. 1 A and IB, the top layer 12 may extend beyond inner layer 14 (when present) and the bottom layer 16 so as to define an overhang portion configured to adhere and/or seal around the edge of the wound. For example, the overhang portion may be adhered to the normal skin (epidermis) surrounding the wound, such as using tissue glue or other adhesives. As shown in FIGS. 1 A and IB, the top layer 12 may have a diameter (D _t ) that is greater than the diameter (D _w ) of the wound. In one embodiment, as discussed below, the top layer 12 may be tailored to the particular wound by shaping its outer edges to conform to the shape of the wound.

[0064] B. Inner Layer

[0065] The inner layer 14 is optionally positioned between the top layer 12 and the bottom layer 16. In one embodiment, the inner layer 14 is composed of a synthetic granulation tissue, such as granulation tissue comprised of endothelial cells, to induce angiogenesis in the wounded area. For example, the synthetic granulation tissue of the inner layer 14 provides a pre-vascularized tissue to augment neovascularization, reduce wound hypoxia, augment cell migration, and/or maintain appropriate progression through the wound healing cycle. Further, fibroblasts may be incorporated within the inner layer 14 to provide cell-based therapy to jump start the later stages of wound healing and provide the substrate for regenerative re- epithelialization of the wound bed.

[0066] The inner layer 14 may be a living layer to help fibroblast and/or keratinocytes from areas surrounding the wound to migrate into the wound

(facilitating healing). In one particular embodiment, the inner layer 14 is composed of a scaffold-free prevascular endothelial-fibroblast construct, SPEC, stem cells, smooth muscle cells, keratinocytes, macrophages, etc., or combinations thereof.

[0067] The inner layer 14 may have a thickness (Ti)of about 0.5 mm to about 2 mm, which is sufficiently thick to provide the benefit of the inner layer 14 while remaining thin enough to allow oxygen diffusion and substrate delivery to support the cellular metabolism and/or removal of waste products. Additional units of this size may be incorporated to address a larger wound bed. [0068] The inner layer 14 may be tailored to the individual, and may be formed from the individual's tissue, such as a skin graft, fat tissue, etc. In other

embodiments, the inner layer 14 may be a living layer from a matched donor's tissue.

[0069] C. Bottom Layer

[0070] The optional bottom layer 16, which will be in contact with the wound, is generally composed of a hydrophilic layer of electrospun collagen fibers. Reaction electrospinning is a technique developed that combines the collagen fibrillogenesis and the traditional electrospinning process to fabricate fibrous collagen mats with diameters that range from nanometers to micrometers.

[0071] In one particular embodiment, native undenatured collagen fibers can be electrospun using benign acidic solvents and reaction electrospinning allowing the collagen to undergo fibrillogenesis during the electrospinning process. Using benign acidic solvents (e.g., such as water and ethanol) at low pH (e.g., about 2 to about 4 using a strong acid, such as HQ), the surface tension can be reduced sufficiently to allow for electrospinning without damaging the protein structure. For example, the collagen can be acidified by adding a strong acid (e.g., HC1) to reduce the pH (e.g., to a pH of about 2 to about 4). Then, the collagen within the strong acid can be diluted using water and the alcohol (e.g., an alkyl alcohol such as ethanol, propanol, isopropanol, butanol, etc.), while keeping the pH relatively low (e.g., a pH of about 2 to about 5, such as about 2 to about 4). The mixture of the water and alcohol can be at a ratio of about 0.5:2 to about 2:0.5 watenalcohol (e.g., about a 1 : 1 ratio). This process can reversibly denature the collagen. Then, the acidic collagen solution can be electrospun within an alkaline atmosphere. The alkaline atmosphere can serve to neutralize the acid mixed with the collagen. In response, the electrospun collagen forms collagen fibers which can be collected within a salt bath. In one embodiment the salt bath is grounded, has a pH of about 7.5 to about 9, and/or includes ammonium sulfate in water. One particularly suitable electrospinning process is described WO 2016/049625 of Yost, et al., which is incorporated by reference herein.

[0072] The porosity of the bottom layer 16 allows the transport of the exudate while providing a moist environment. The porosity of the bottom layer 16 may be varied as desired with respect to the particular wound being treated. In certain embodiments, the bottom layer 16 may have an average void size of about 10 micrometers (micron, μιη) to about 100 μιη through the bottom layer 16.

[0073] In one embodiment, the bottom layer 16 includes an active agent (P), such as an anti-inflammatory drug, an antipurinergic peptide, a peptide tailored to the particular wound's needs, etc., or mixtures thereof. For instance, the wound may be biopsied to determine any lacking material, and then that bottom layer 16 may be tailored to include material within the electrospun collagen fibers to address the deficiency diagnosed from the biopsy. Thus, the wound dressing 10 may be designed uniquely for the individual wound being treated.

[0074] Any disruption of the tissue will illicit an inflammatory response. Any materials for blocking or attenuating these responses may be utilized in the bottom layer 16 to control the inflammatory response within the wound. For example, IL- lRa CYT-658 may be utilized as an inhibitor of the IL1 receptor (e.g., 0.5 ng/ml, IL- lRa CYT-658 in PBS). In another example, mefloquine or suramin may be utilized as broad channel blockers to inhibit purinurgic signaling. As another example, C34 (i.e., 1 -Methyl ethyl 2-(acetylamino)-2-deoxy-a-D-glucopyranoside 3,4,6-triacetate) may be utilized to block the TLR4 receptor, if desired in the particular wound.

Additionally, complement inhibitors may be used to address the prolonged inflammatory response including CR2-Crry, Crry-Ig, anti-C5 mAb, C3a Receptor, C5a Receptor Antagonist (RA), CR2-fH, anti-fB, CR2-CD59, etc., or combinations thereof.

[0075] In one particular example, the bottom layer 16 may be coated with an antiinflammatory peptide such as a therapeutic connexin43 mimetic peptide (JM2). In such a system, a detrimental innate inflammatory response is muted using connexin- based peptidomimetic inhibitors. Connexins are proteins that form channels in the membrane that allow for diffusion of small molecules between cells, and between the inside and outside of cells. Connexin 43 (Cx43) expression increases in blood vessels in response to tissue injury. The extracellular ATP release has been shown to mediate early inflammatory responses. JM2 is a peptide developed to target the microtubule binding domain of Cx43, and has been shown to regulate the inflammatory response. This unique peptide attenuates the endothelial ATP release, dampening the neutrophil response, decreasing the early inflammatory response. [0076] The concentration of the anti-inflammatory peptide may vary based on the diagnosis of the deficiencies of the wound. The concentration of the antiinflammatory peptide within the bottom layer may be varied as desired with respect to the particular wound being treated. In certain embodiments, the bottom layer may have an average concentration of the anti-inflammatory peptide in the range of about 25 micromolar to about 250 micromolar (10-6 mol/L).

[0077] As another example, wounds with relative IL-6 deficiency or

proangiogenic factors (such as platelet-derived growth factor, PDGF) could be treated specifically by including the factor(s) to the electrospun collagen layer. In one embodiment, wounds with high propensity towards infection could be treated with an anti-microbial/myotic/viral specifically included in the electrospun collagen layer.

[0078] The bottom layer may be tailored to the particular wound, in terms of thickness (T _b ), shape, and/or size (D _t ). That is, the electrospun collagen of the bottom layer may be formed to any desired thickness across the wound dressing.

Additionally, the inner cellular layer can be designed to span the entirety of the wound bed, as well as the two components of the top layer. For example, all layers of the device may be tailored to the size of a specific wound.

[0079] II. Method of Forming Wound Dressing

[0080] The wound dressing 10 of FIGS. 1A, IB, 2A, and 2B may be formed, in one embodiment, from the top layer 12 down. For example, the fibrous sheet of the top layer 12 may be coated (e.g., sputter-coated) on one side with silver particles, and then an optional inner layer 14 may be formed on the opposite side of the fibrous sheet of the top layer 12.

[0081] In one embodiment, the inner layer 14 may be formed by culturing cells from the patient (e.g., endothelial cells and/or fibroblast) directly on the surface of the fibrous sheet of the top layer 12 that is opposite from the silver coated surface 13. In certain embodiments, an adhesive layer can first be coated on the fibrous sheet so as to be positioned between the fibrous sheet and the inner layer. For instance, the adhesive layer may include fibronectin as an adhesion layer for the cells of the inner layer.

[0082] Finally, a bottom layer 16 may be attached, either directly to the top layer 12 or to the inner layer 14 (when present). If the inner layer is not present, then the bottom layer may be formed by electrospinning collagen fibers directly onto the surface of the fibrous sheet of the top layer that is opposite from the silver coated surface. Alternatively, if an inner layer of living cells is present, then the bottom layer may be formed separately and then laid onto the inner layer.

[0083] III. Method of Treating a Wound

[0084] As stated above, the wound may by scanned to determine its shape and depth prior to applying the wound healing matrix thereto. For example, FIG. 7 shows a wound 100 in a patient 102. An imaging device 104 may detect the outer dimensions of the wound 100 to form the diameter (D _w ) of the wound 100, which can be translated to the diameter (D _w ) of the bottom layer and the inner layer (when present). Additionally, the imaging device 104 may detect the outer dimensions of the wound 100 to form the diameter (D _t ) of the top layer to adhere the wound dressing to the skin 101 of the patient 102. Finally, the imaging device 104 may detect the inner dimensions of the wound 100 to determine the depth of the wound which may be varied across the wound 100.

[0085] The imaging device 104 may communicate the image to a computing device 106, which may include one or more processor(s) and one or more memory device(s). The one or more processor(s) can include any suitable processing device, such as a microprocessor, microcontroller, integrated circuit, logic device, and/or other suitable processing device. The one or more memory device(s) can include one or more computer-readable media, including, but not limited to, non-transitory computer-readable media, RAM, ROM, hard drives, flash drives, and/or other memory devices. The one or more memory device(s) can store information accessible by the one or more processor(s), including computer-readable instructions that can be executed by the one or more processor(s). The instructions can be any set of instructions that when executed by the one or more processor(s), cause the one or more processor(s) to perform operations. In some embodiments, the instructions can be executed by the one or more processor(s) to cause the one or more processor(s) to perform operations, such as any of the operations and functions for which the computing devices are configured, such as the operations for forming a computer image in a manageable format (e.g., computer-aided design or "CAD" format). The instructions can be software written in any suitable programming language or can be implemented in hardware. Additionally, and/or alternatively, the instructions can be executed in logically and/or virtually separate threads on processor(s). The memory device(s) can further store data that can be accessed by the one or more processor(s). For example, the data can include models, formulas, etc. that can be used to shape the bottom layer of the wound dressing to substantially match the shape of the wound 100 detected. The data can also include other data sets, parameters, outputs, information, etc. shown and/or described herein.

[0086] The computing device(s) also includes a communication interface used to communicate, for example, with the electrospinning device 108. The communication interface can include any suitable components for interfacing with one or more network(s) or electronic components, including for example, transmitters, receivers, ports, controllers, antennas, and/or other suitable components. Communication interface can be used to communicate with other electronic devices over one or more networks, such as e.g., a local area network (LAN), a wide area network (WAN), SATCOM network, VHF network, a HF network, a Wi-Fi network, a WiMAX network, a gatelink network, and/or any other suitable communications network for transmitting messages to and/or from the imaging device 104 or the electrospinning machine 108.

[0087] As such, the electrospinning machine 108 may form the bottom layer to a shape and thickness that corresponds to the wound 100, and may include varying thickness across the wound dressing through varying the thickness of the bottom layer.

EXAMPLES

[0088] This study describes the development and characterization of the top layer of the wound dressing. Results indicate silver coated 30kDa membranes retained their filtration capabilities, while silver coated lOOkDa membranes did not. However, with the example described herein, the top coating appears to be toxic to cells.

[0089] Ultracel Regenerated Cellulose Ultrafiltration Membranes with filtration capacities of lOOkDa and 30kDa and diameters of 25mm were purchased from EMD Millipore. The membranes were immersed in a 10% Ethanol solution for 72 hours. Following the 72 hour time period, the membranes were placed on glass slides. The glass slides containing membranes were then placed on a slide warmer, allowing any remaining moisture to evaporate from the membranes so that they could dry. After drying, the membranes were organized and stored based off of their filtration capacities. When ready for silver deposition, the membranes were placed in Whatman Filter Holders to physically secure them. After preparing these membranes for silver deposition, a silver target was loaded into a Denton Vacuum Desk V HP sputter coater. Then, the Whatman Filter Holders containing a membrane were placed in the sputter coater. For characterization of the silver coated membranes, the following silver deposition parameters were selected on the sputter coater: 30mAmp@150s, 25mAmp@125s, and 20mAmp@100s. Only one parameter was used per membrane and each membrane was sputter coated individually.

[0090] Fig. 2 is a picture of a membrane after silver deposition. Following silver deposition, the membranes were stored in either a Whatman Filter Holder or a 35mm petri dish for characterization.

[0091] Microscopy

[0092] Three 30kDa and three lOOkDa membranes were prepared and sputter coated using the protocol and parameters described above. Fig. 3 A is a picture of a woven polyester membrane after silver deposition. Following silver deposition, the membranes were stored in either a Whatman Filter Holder or a 35mm petri dish for characterization. Fig. 3B is a picture of a hydrophilic PTFE membrane without silver deposition. One 30kDa and one lOOkDa membrane with no preparation or silver deposition was used as a control. Then, the silver coated membranes were examined under a microscope. Pictures were taken at a magnification of 200x.

[0093] Microscopic analysis reveals the silver particles adhered directly to the cellulose fibers. This quality is beneficial because selective filtration of the developed product is desired. The cellulose fibers appear lighter with the decreasing current and time of each sputter coating parameter. This indicates that silver deposition is highest on membranes with a 30mAmp@150s sputter coating parameter and lowest on membranes with a 20mAmp@100s sputter coating parameter.

[0094] The silver deposited and adhered directly to the cellulose fibers of the membrane. Since an ultrafiltration capability is desired, this characteristic is beneficial for the future development of the wound dressing. [0095] Contact Angle

[0096] In order to characterize the hydrophobic nature of the silver coated membranes, contact angle analysis was performed as described in (Bracco, G., & Hoist, B. (2013). Surface science techniques. Heidelberg: Springer.). First, woven polyester membranes were prepared and sputter coated using the protocol and parameters described above. A piece of plastic with a known length of 6.2mm was placed on the membrane. Then, 2μΙ. of a solution containing distilled water and blue dye was pipetted onto the membrane. Four 2[iL drops of water were pipetted onto each membrane. Pictures of each membrane were taken. This was performed two times per membrane to ensure that there would be a ten 2[iL drop total. The pictures were uploaded into cell Sens imaging software and contact angle measurements were taken using the following equation: CA=2 tan ^A (-l) [(drop height)/(drop radius)] .

[0097] Contact angle analysis shows that the silver coated membranes are very hydrophobic.

[0098] FIGS. 4A and 4B show the contact angle of silver-coated woven polyester.

[0099] Toxicity

[00100] Three woven polyester membranes were prepared and sputter coated using the protocol and parameters described above, three for each parameter. Three plain woven polyester membranes (i.e., no silver-coating) were used as control. The membranes were sterilized and placed in a 35mm petri dish. 2mL of sterile filtered Simulated Body Fluid was added to submerge each membrane. Following inoculation, the silver coated membranes were incubated at 37°C. On days 2, 8, and 14, approximately 2mL of SBF was transferred into 15mL conical tubes and stored at 4°C. After all time points of SBF were collected, endothelial cells were cultured to confluence. A 96-well plate was inoculated with endothelial cells with approximately three thousand endothelial cells per well. The 96-well plate was then incubated for 24 hours at 37°C. Following incubation, each well was inoculated with ΙΟΟμΙ. of endothelial cell media, EGM2(Lanza), and ΙΟΟμΙ. of SBF collected from days 2,5, and 7. For this experiment, five controls were chosen: control 1 contained endothelial cells with ΙΟΟμί of stock SBF and ΙΟΟμΙ. of EGM2, control 2 contained endothelial cells with 200μί of EGM2, control 3 contained 200μΙ. of stock SBF, control 4 contained ΙΟΟμΙ. of stock SBF and ΙΟΟμΙ. of EGM2, control 5 contained 200μΕ of EGM2. The 96-well plate was then incubated for 24 hours at 37°C. After incubation, each well was inoculated with 20μΙ. of AlamarBlue, excluding controls 4 and 5. The 96-well plate was then incubated for 3 hours at 37°C, allowing the cells to metabolize the blue non fluorescent dye to red fluorescent resorufin. Following incubation, fluorescence measurements were taken with a microplate reader using and excitation length of 535 nm and emission of 590 nm. This was performed in triplicate.

[00101] As indicated in Fig. 6, more than half of the cells were alive with the all parameters.

[00102] FIGS. 8 A and 8B show a picture of the wound dressing according to these examples. FIGS. 9A and 9B show a scanning electron micrograph image of reaction electrospun collagen fibers, and FIG. 9B a reaction electrospun fibers seeded with endothelial cells and fibroblasts. The endothelial cell network is shown, with the nucleus and cytoskeleton. FIGS. 10A, 10B, and IOC show the tissue engineered living pre-vascular network composed of 4: 1 fibroblasts to endothelial cells, SPEC. FIG. 10A shows the honeycomb shape that was selected for our initial experiments. This shape allows granulation tissue to invade and help integrate this portion of the SWOD into the host, scale bar is 1 mm. FIG. 10B shows the endothelial cell network within the fibroblast-ECM support structure, with the Cytoskeleton and CD31. FIG. IOC is an H&E stain of the SPEC 24hrs post implantation in a Sprague Dawley rat. Endothelial lined vascular structures are present that contain red blood cells.

[00103]

[00104] These and other modifications and variations to the present invention may be practiced by those of ordinary skill in the art, without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. In addition, it should be understood the aspects of the various embodiments may be interchanged both in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the invention so further described in the appended claims.