WO2017035280A1 - Skin damage healing aids and dressings - Google Patents

Abstract

The device of the invention includes dressings having a cathode and an anode, each forming an electrical half-cell, and a conductive treatment gel containing HADSCC media.The treatment gel may also contain human adipose-derived stem cells. The method includes providing a treatment gel and a dressing, applying the treatment gel to the skin or to the dressing, and applying the dressing to the skin. Other embodiments include bandages combining sterile dressings with a composition including a conditioned medium. The conditioned medium may be nanoencapsulated or may be dispersed in an emollient base and applied to the sterile dressing.

Description

TITLE: Skin damage healing aids and dressings

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to US Provisional Application 62/209319 filed 24 August 2015 and to US Provisional Application 62/209324 filed 24 August 2015, the disclosures of which are incorporated by reference for all purposes.

INVENTORS: Sanjay Dhar, Mohammad Ali Kharazmi, and Mohammad Saeed Kharazmi

APPLICANT: NuGene, Inc.

BACKGROUND OF THE INVENTION

Field of the Invention

[002] This invention is in the field of dermal care and treatment. In particular, it concerns compositions that improve the condition of skin damage by topical application together with dressings Skin damage includes burns, wounds, scars, wrinkled skin, blemished skin, acne, or other skin conditions. The invention also includes methods of treating skin using such compositions and dressings. In embodiments, the dressings may include electroactive dressings.

Wounds and skin damage

[003] Skin may be damaged by a variety of chronic insults including aging, sun exposure, past wound healing, and cosmetic treatments. In particular, as skin ages, circulation lessens and dermal fibroblast cells become less active; skin becomes thinner and loses elasticity. Aged skin shows a decrease in volume and elasticity. There is a need to prevent and reverse these conditions to maintain healthy, youthful skin.

[004] Skin may be damaged by other more acute conditions such as infections, wounds, and acne. There is a need to treat these conditions that accelerates healing while regaining the appearance and resiliency of healthy skin.

Human adipose-derived stem cells

[005] A stem cell is an undifferentiated or relatively undifferentiated cell that is capable of giving rise to more cells of the same type, and from which certain other kinds of cell arise by differentiation. There are a variety of human stem cells that serve as reservoirs for recovery and replacement of damaged tissue. Adipose-derived stem cells are stem cells extracted from adipose tissues. Human adipose tissue is available ex vivo as a result of cosmetic procedures including liposuction. Adipose tissue, like other tissue types, is not a homogenous mixture of a single cell type. Instead adipose tissue includes a combination of fat cells, vasculature, connective tissue, and blood cel ls. Stem cells may be extracted from such tissue by any of a number of methods known in the art, including treatment with su rfactants or enzymes (including proteases such as such as collagenase, or trypsin), maceration, separation by centrifugation, filtering, or settling, ultrasonic treatment, adherent culturing, or some combination of these methods.

[006] Once extracted from adipose tissue, adipose-derived stem cells may be grown in tissue culture by a number of methods known in the art, including growth on three-dimensional scaffolds or supports, growth in suspension culture, or growth on the surface of plastic or glass vessels.

[007] Culture of cells for human use must take place under sterile conditions with particular attention paid to the avoiding contamination by infectious organisms, including those infecting the stem cell donors. Contamination may also arise from culture materials: for example, use of serum products, such as bovine fetal calf serum, is very common in cell culture. Such products may include infectious agents or antigenic or allergenic materials. One object of the invention is to provide treatment compositions derived from human stem cells that have not been cultured in the presence of or exposed to serum products.

[008] Human adipose-derived stem cells and their progeny produce a variety of growth-promoting and healing materials such as growth factors, cytokines, stress proteins, and nutrients including TGF-B, PDGF, and GM-CSG, interleukins, and matrix proteins (collectively, stem cell products). While many of these have been identified, the cells likely also secrete other substances due to their pluri-potency either not yet known or with beneficial functions yet to be precisely identified. Some of these materials may be effective at low concentration. Another object of this invention is to provide treatment compositions including stem cell products.

[009] Autologous human adipose derived stem cells are stem cells derived from adipose tissue of an individual and subsequently used in treating that individual.

[0010] Tissue-derived products, particularly those derived from minimally processed human tissue, may contain human infectious agents or antigens. The invention includes embodiments with

compositions containing stem cells autologous with the treated individual. Such compositions are not necessarily free of infectious agents or antigens, but the treated individual has already been exposed to these autologous agents and antigens so that the risk of further infection or immune reaction should be low. Another object of this invention is to provide treatment compositions including autologous stem cells to aid healing with reduced risk of infection or immune reaction. Human adipose-derived stem cell conditioned ("HADSCC") medium

[0011] Human stem cells, such as adipose-derived stem cells, produce a variety of growth- promoting and healing materials such as growth factors and cytokines. These mixtures of stem cell products may be harvested from cultured adipose-derived stem cells by collecting the culture medium to which such cells have been exposed.

[0012] Growth of such adipose-derived stem cells includes supply of nutrients for the cells through provision of an aqueous culture medium. Cells grow in culture in contact with medium and extract nutrients from it. These cells also deliver to the medium products of their growth and metabolism. Among the products are the growth factors and cytokines discussed above as well as metabolic products. Conventional tissue culture requires replacement of culture medium as cells use up nutrients and deliver products that may affect future cell growth. This replacement may be either continuous, with a portion of the medium removed as new medium is added, or intermittent with periodic replacement of some or all of the culture medium in a vessel. Culture medium removed after exposure to cells in culture is known as spent or conditioned medium. Culture medium removed after exposure to human adipose-derived stem cells will be referred to as HADSCC medium.

Stem cells, media, dressings, and electric fields in wound healing

[0013] Numerous publications report that application of exogenous tissue-derived cells or stem cell products enhances healing of skin wounds. For example, Ebrahimian ef al. report in Arteriosclerosis, Thrombosis, and Vascular Biology 29, 4, 2009; pp 503-510 that adipose tissue-derived stroma cells participate in dermal wound healing in physiological and pathological conditions by their ability to promote reepithelialization and angiogenesis. Exogenously applied cells improved skin blood perfusion, promoted dermal wound healing and enhanced wound closure, viscoelasticity, and collagen tissue secretion. The authors concluded that adipose lineage cells represent a new cell source for therapeutic dermal wound healing. PCT US2014/034738, US provisional application 62/112124, and US provisional application 62/154120, all commonly assigned with this application, describe a variety of compositions containing HADSCC media suitable for wound, scar, burn, and damaged skin treatment.

[0014] Many tissues, including living layers of the skin, respond to appropriate mixtures of growth factors to encourage regeneration. For example, US 2012/0065129 Al to Park ef al. asserts that a culture medium of adipose-derived stem cells and growth factors isolated from the culture medium can be advantageously applied in drugs, quasi drugs, and cosmetics for wound healing. PCT US2014/034738, commonly assigned with this application, describes dermal treatment compositions including a med ium recovered from human adipose-derived stem cell culture (HADSCC).

[0015] Bandages for wounds or burns are commonly composed of sterile absorbent dressings that are fastened in place by separate fasteners such as tape, adhesives, compressive textiles, or ties. Some bandages may be pretreated with antimicrobials to retard wound infection. For example, wound dressings impregnated with certain healing promoting or microbiocidal materials, such as nanosilver, cause wounds to heal more quickly. Nanosilver is used extensively in wound management, particularly in burn treatment. Other reported applications include chronic wounds, burns injuries in children and neonates, ulcers (including diabetic ulcers, rheumatoid arthritis-associated leg ulcers, and venous ulcers), toxic epidermal necrolysis, healing of donor sites, and meshed skin grafts. Other bandages may be untreated but applied with or over topically applied aids such as antimicrobials, clotting factors, desiccants.

[0016] US2012/0318262 to Lee ef al. describe that dressings that may include materials such as pyrvinium or its salts or analogs, gelatin, silver, cellulose, alginate, collagen, a hydrocolloid, a hydrogel, a skin substitute, a wound filler, a growth factor, an antibody, a protease, a protease inhibitor, an antibacterial peptide, an adhesive peptide, a hemostatic agent, living cells, honey, nitric oxide, a corticosteroid, a cytotoxic drug, an antibiotic, an antimicrobial, an antifungal, an antiseptic, nicotine, an anti-platelet drug, an NSAID, colchicine, an anti-coagulant, a vasoconstricting drug or an

immunosuppressive.

[0017] US 2011/0091568 to Lipton ef al. describes several types of stem cells and media derived from growth of those cells. Lipton also discloses "plasters, bandages, dressings, gauze pads, patches and the like, containing an appropriate amount of conditioned medium and, optionally, other ingredients. In some cases use may be made of plasters, bandages, dressings, gauze pads, patches and the like which have been impregnated with a topical formulation containing the therapeutic formulation." Lipton ef al. however does not describe HADSCC medium, does not describe "an appropriate amount" other than "optimal percentage of the conditioned media or extract in each composition can vary according to the composition's formulation and the therapeutic effect desired" and by reciting a range of concentrations (apparently in relation to cosmetic procedures) that span more than six orders of magnitude. Lipton ef al. also does not describe any details which would enable the skilled practitioner to produce effective plasters, bandages, dressings, gauze pads, patches and the like. There is thus a need for a bandage that contains HADSCC medium in proportions and amounts that support effective healing.

[0018] US 8911963 to Epstein ef al. describe a therapeutic bandage including conditioned medium and/or processed conditioned medium impregnated into a polymer. The therapeutic bandage is a polymer impregnated with processed conditioned medium that is applied topically in the form of a bandage, patch, or a configured mesh. An embodiment of the bandage is described as a gauze bandage impregnated with a biodegradable polymer comprising conditioned medium. The bandage can be lyophilized and stored for future use, then reconstituted by added moisture or by wound moisture. The polymer is said to permit sustained release of the processed cond itioned medium. In other

embodiments, the polymer may be a non-biodegradable polymer that removed after use. The stem cells can be isolated from adipose tissue. The described therapeutic bandages rely on the polymer embedding to retain and release an effective mix of growth factors from the bandage over an effective time course. HADSCC medium contains a complex mix of growth factors that may not be equally soluble in a given polymer. There is thus a need for a bandage that contains HADSCC medium where retention and release of growth factors does not rely on polymer embedding.

[0019] Numerous publications also report that healing of skin wounds is stimulated by electrical current. See, for example, OM Alvarez in J Investigative Dermatology 81, 1983; pp.144-148. TA Banks, ef al. reported in Integr. Biol., 2015; 7, pp. 693-712 that human bone marrow-derived mesenchymal stem cells migrate in response to applied electric fields. They remarked regarding the significant regenerative potential in the observed improved healing in vivo post applied electric fields and that the intrinsic piezoelectric nature of collagenous-rich tissues, such as bone and cartilage, can result in the production of small, endogenous electric fields (EFs) during applied mechanical stresses. HH Park ef al. reported in Appl. Phys. Lett. 105, 2014; 24, p 4109 that induced electric fields could control directional migration of rat mesenchymal stem cells. The authors observed mesenchymal stem cell migration during wound closure in presence of an indirect electric field. B Vanhaesebroeck in Nature Chemical Biology 2, 2006; pp. 453 - 455 reported that manipulation of electric fields affect wound healing in vivo and identified the phosphoinositide 3-kinase signaling pathway as a key component of cell migration in response to electric cues.

[0020] Electroactive wound dressings produce local electric fields by providing electrical half cells in proximity to healing skin. Wound exudate or exogenously administered fluid close the half cells into a full electrical cell, or battery of electrical cells that generate low-level currents between electrodes on the dressing and extending into proximal healing tissue. Commercially available dressings sold by Vomaris Wound Care, Inc. of Tempe Arizona under the registered trademark Procellera^® are said to provide effective antimicrobial protection to the wound site, inhibiting the growth of harmful microorganisms that may cause infection. Without infection, wounds are said to heal faster. The dressings feature a staggered matrix pattern silver or silver chloride and zinc electrodes dots applied to the dressing surface.

[0021] The range of penetration of the current produced by electric fields from electroactive dressings depends on the conductive path between the tissues and the dressing, upon the configuration of the tissues themselves, and upon the pattern of electrodes on the dressings. There is a need for a dressing that provides a pattern that alters the field distribution as compared to conventional dressings to provide some measure of targeting to the current produced. There is also a need for a dressing and composition that provides stem cell products or even stem cells themselves at the same location as the current.

[0022] Thus exogenous stem cells (and media derived from culture of such cells) su pport wound healing; wound healing is to some measure dependent on natural electric fields in tissue; and exogenously applied electric fields can promote both cellular migration and wound healing. Half-cell arrays on wound dressings can deliver electric fields to healing wounds and skin. There is however a need to provide a wound dressing and composition that exploits synergistic combinations of these effects to further promote healing and restoration of the appearance of healthy skin.

BRIEF SUMMARY OF THE INVENTION

[0023] In embodiments, the invention includes kits, compositions, and methods. The kits of the invention include a dressing having a cathode and an anode each forming an electrical half-cell and a conductive treatment gel containing HADSCC media. The gel also may include a gelling agent and a viscosity of at least 3000 cP. In some embodiments, the viscosity may range between about 5000 and about 20000cP. The dressing may further include a flat flexible substrate having parallel first and second su rfaces. The first surface may support the anode and the cathodes. The anode may include metallic zinc deposited on a region of the first surface of the substrate and the cathode may include metallic silver deposited on a second region of the first surface of the substrate. The first region does not overlap the second region. The first region may include a plurality of discrete spots distributed in a regular array. The second region may also include a plurality of discrete spots distributed in a regular array, with the first region spots intercalated among the second region spots. In other embodiments, the first region includes a plurality of discrete spots, and the second region includes a plurality of circles, each circle enclosing one of the spots.

[0024] The treatment gel may include HADSCC medium of at least 50% by weight and gelling agent of at least 0.5% by weight of the treatment gel. In embodiments, the gelling agent may be at least 1% by weight of the treatment gel. The gelling agent may be a hydroxymethyl cellulose or a carboxymethyl cellulose. The treatment gel may include ionic salts from about 50 mEq/L to about 200 mEq/L and in some embodiments about 140 mEq/L. Components of the gel from any of these ranges may be combined with other components present in any of their respective ranges.

[0025] In other embodiments, the invention includes a conductive treatment gel containing human adipose-derived stem cells for topical application. The treatment gel may contain a gelling agent of at least 0.5% by weight and a culture medium to support the cells. The gelling agent may be a

hydroxymethlyl cellulose or a carboxymethyl cellulose and the gel may have a viscosity of at least 3000 cP. In some embodiments, the viscosity may range between about 5000 and about 20000cP. The composition may include ionic salts from about 50 mEq/L to about 200 mEq/L and in some

embodiments about 140 mEq/L. The treatment gel may further include a culture medium, which may include one of Dulbecco's Modified Eagle's Medium or RPMI 1640 medium. The medium may contain 0.0025 - 0.5% nanosilver particu late by weight of the medium. Components of the gel from any of these ranges may be combined with other components present in any of their respective ranges.

[0026] The invention also includes a method of treating a skin condition in a human. The method includes the steps of providing a treatment gel and a dressing having complementary electrical half-cells, applying the treatment gel to the skin or to the dressing, a nd applying the dressing to the skin. The dressing includes a first region and a second region distinct from the first region, with the first region having a metallic zinc deposit and the second region having a metallic silver deposit. The first region may include a first plurality of discrete spots, and the second region may include a second plurality of discrete spots. The first plurality of discrete spots may be intercalated among the second plurality of discrete spots, forming a matrix of alternating zinc and silver spots. Alternatively, first region may include a plurality of discrete spots, and the second region may include a plurality of circles, with each circle enclosing one of the spots. The spots may be separated by about 2 mm or less. The treatment gel has a viscosity of at least about 3000 cP and an ionic content of between about 50 mEq/L to about 200 mEq/L and in some embodiments about 140 mEq/L. In some embodiments, the viscosity may range between about 5000 and about 20000cP. The treatment gel may contains an HADSCC medium. The HADSCC medium may form at least 50% by weight of the treatment gel. In some embodiments the gel may contain 0.0025 to 0.5% nanosilver particulate by weight of the medium.

[0027] In other embodiments of the method, the treatment gel contains human adipose-derived stem cells autologous to the human receiving the treatment. The treatment gel may further include a culture medium, which may include one of Dulbecco's Modified Eagle's Medium or RPMI 1640 medium. In some embodiments the gel may contain 0.0025 to 0.5% nanosilver particulate by weight of the medium. The skin condition may include one or more of a wound, a scar, a blemish, acne, a stretch mark, or a wrinkle.

[0028] In some embodiments, the invention includes a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be a sterile dressing with the conditioned medium applied to the sterile dressing. The conditioned medium may be contained in nanocapsules, and may be concentrated by centrifugal filtration prior to loading into the nanocapsules. The nanocapsules may be applied to the sterile dressing as an aqueous suspension and air dried. The nanocapsules may include nanoliposomes or polymer nanocapsules.

[0029] In other embodiments the invention includes a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be dispersed in an emollient base and the emollient base applied to the sterile dressing. The conditioned medium may be harvested from culture of human adipocyte-derived stem cells. The conditioned medium may form about 40% by weight of the composition.

[0030] The stem cells may be cultured in Dulbecco's Modified Eagle's Medium or RPMI 1640 medium containing 0.0025 - 0.5% of a nanosilver particulate by weight. The nanosilver particulate includes a stabilizing coating including one or more of citrate, tannic acid, polyvinylpyrrolidone, silica, polyethylene glycol, oligonucleotides, or a peptide. The emollient base includes an oil and an emulsifying agent, with the oil forming about 5-20% by weight of the composition and the emulsifying agent forming about 10-20% of the composition. The oil may include one or more of coconut oil, avocado oil, neem oil, rosemary oil, manuka oil, safflower oil, or geranium oil.

[0031] In some embodiments, the sterile dressing includes a textile or a polymer. In other embodiments, the sterile dressing is a film, foam, semi-solid gel, pad, gauze, or fabric. The bandage may also include a substance or element for the fixation of the bandage to a wound, such as a tie, an adhesive, a tape, a compression stocking, or an adherent gel. The sterile dressing may have a wound- facing surface with a peripheral area and a central area. The peripheral area may include an adhesive, and the emollient base may be applied to the central area. In other embodiments, the sterile dressing includes a wound-facing surface, and the wound-facing surface includes an adhesive over substantially the entirety of the wound-facing surface. The sterile dressing includes a second surface opposing the wound-facing surface, and the emollient base may applied to the second surface. [0032] The invention also includes a method including the steps of applying to a wound a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be dispersed in an emollient base and the emollient base applied to the sterile dressing. The bandage is then secured to the wound. In some embodiments, the method also includes adding a liquid to the bandage before applying the bandage to the wound. In some embodiments the composition is dried and the liquid rehyd rates the dried composition. In other embodiments, the emollient base su bstantially immobilizes the conditioned medium and the liquid elutes the conditioned medium from the bandage. The wound may be any of a dermal wound, an epidermal wound, a burn, an infectious lesion, a surgical site, an ulcer, or a scar.

[0033] In other embodiments, the invention also includes a bandage having a composition applied to a sterile dressing. The composition has major components of about 5-30% by weight of an oil, about 10-30% by weight of an emulsifying agent, and about 40-60% by weight of a conditioned medium harvested from culture of human adipocyte-derived stem cells. The composition may also include about 1-5% vitamin E or coconut oil.

[0034] In embodiments the invention includes bandage that includes a sterile dressing and a conditioned medium applied to the sterile dressing. The conditioned medium may be concentrated from media harvested from culture of human adipose derived stem cells. The conditioned media may contain nanosilver.

[0035] In any of the embodiments described, the sterile dressing may include a flat flexible su bstrate having a first surface and a second surface parallel to the first surface. The first surface may su pport a cathode and an anode. The anode may include metallic zinc deposited on a first region of the first surface of the substrate and the cathode may include metallic silver deposited on a second region of the first surface of the substrate. The first region does not overlap the second region. The first region may include a first plurality of discrete spots, and the second region may include a second plurality of discrete spots. The first plurality of discrete spots may be intercalated among the second plurality of discrete spots to form a regular array.

[0036] In other embodiments, the invention includes a skin damage treatment device including a dressing and a conductive treatment gel. The dressing has a cathode and an anode. Each of the cathode and the anode form an electrical half-cell, The treatment gel has a human adipose-derived stem cell culture ("HADSCC") medium and a gelling agent. The treatment gel has a viscosity of at least 3000 cP and ionic salts in the range of about 50 mEq/L to about 200 mEq/L. [0037] The dressing further includes a flat flexible substrate having a first surface and a second su rface parallel to the first surface. The first surface supports the cathode and the anode, and the cathode and the anode include a first electrode and a second electrode. The second electrode forms a closed ring surrounding the first electrode.

[0038] The embodiments described may form the anode from a plurality of discrete anode spots. The cathode includes a plurality of discrete cathode spots; the plurality of anode spots may be intercalated among the plurality of cathode spots.

[0039] The treatment gel may include at least 50% by weight of the HADSCC medium and at least about 0.5% by weight of a gelling agent. In some embodiments, the treatment gel includes at least 1.0% by weight of the gelling agent, and the gelling agent includes a hydroxymethyl cellulose or a

carboxymethyl cellulose.

[0040] In other embodiments, the invention includes a conductive treatment gel including a gelling agent of at least 0.5% by weight, a human adipose-derived stem cell, a culture medium, and ionic salts at a concentration of about 50 mEq/L to about 200 mEq/L.

[0041] The gelling agent may include a hydroxy methly I cellulose or a carboxymethyl cellulose and the gel may have a viscosity of at least 3000 cP. The culture medium may include about 0.0025 to about 0.5% nanosilver particulate by weight.

[0042] The invention also includes a method of treating a skin condition in a human having steps of providing a treatment gel and a dressing. The dressing includes complementary electrical half-cells. The method has further steps of applying the treatment gel to the skin or to the dressing, and applying the dressing to the skin to be treated. The gel contains one of a human adipose-derived stem cell conditioned medium or a human adipose-derived stem cell.

[0043] In embodiments of the above method the gel may contain human adipose-derived stem cells autologous to the human, a culture medium, and about 0.0025 to about 0.5% nanosilver particulate by weight of the medium.

[0044] The invention also includes embodiments of a bandage having a sterile dressing; and a composition with a conditioned medium applied to the sterile dressing. The conditioned medium may be contained in a plurality of nanocapsules. The nanocapsules may be applied to the sterile dressing as an aqueous suspension and air dried. The aqueous suspension may include a nanosilver particulate.

[0045] The conditioned medium may be dispersed in an emollient base with the emollient base applied to the sterile dressing. The emollient base may include an oil and an emulsifying agent with the oil forming about 5-20% by weight of the composition and the emulsifying agent forming about 10-20% of the composition. The conditioned medium may be harvested from culture of human adipocyte- derived stem cells and may form about 40% by weight of the composition.

[0046] In other embodiments, the invention also includes a method of treating a wound including steps of applying the bandage as described above to the wound and securing the bandage to the wound. In the method, the composition may be dried and the method also includes adding a liquid to the bandage to rehydrate the composition.

[0047] In still other embodiments, the invention includes a bandage having a sterile dressing and a composition consisting essentially of about 5-30% by weight of an oil, about 10-30% by weight of an emulsifying agent, and about 40-60% by weight of a conditioned medium harvested from culture of human ad ipocyte-de rived stem cells.

[0048] In other embodiments, the invention includes a bandage having a sterile dressing and a conditioned medium applied to the sterile dressing. The conditioned medium is concentrated from media harvested from culture of hu man adipose derived stem cells and includes a nanosilver particulate.

[0049] Any of the described bandages may include a sterile dressing have a flat flexible substrate with a first surface and a second surface parallel to the first surface. The first surface supports a cathode and an anode. The cathode and the anode include a first electrode and a second electrode, and the second electrode forming a closed ring surrounding the first electrode.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0050] Fig. 1 illustrates a prior art dressing including an array of cathodes and anodes.

[0051] Fig. 2 illustrates an embodiment of a dressing of the invention that includes an array of core- ring electrodes.

[0052] Fig. 3 illustrates steps of an embodiment of a method of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0053] A large number and variety of materials are useful in forming wound dressings. A wound dressing typically includes a film, gel, foam, gauze, textile, polymer, or fabric. Some dressings may include non-absorbable components such as silicone, polyacrylamide gels, conventional textiles, or PTFE. Other dressings, particularly those used for internal wounds, may include polymers that are naturally biodegradable in the body such as fibrin, PLGA, PGA, PLA, polycaprolactone or hyaluronic acid. Dressing may be designed to adhere or to remain free of the wound surface.

[0054] Dressings may be supplemented by creams, ointments, or included materials that promote healing or reduce infection. This invention includes compositions applied to the dressings themselves that promote healing of skin damage to which the dressings are applied.

[0055] Appropriate wound closure methods depend on wound severity. Staples or sutures require nurse of physician attendance and sometimes require an anesthetic. Many of these devices leave scars, including scars beyond the wound itself from insertion holes. Varying tensions applied to a laceration or surgical incision between the suturing points cause skin or tissue puckering, increasing the appearance of scars. These skin closure methods may also require follow-up visits to a clinic for removal of sutures or staples.

[0056] Less severe wounds may be closed by over-the-counter adhesive-based dressings and more complex assemblages of gauze, creams, ointments, tapes, or applied vacuum. Covering a wound with an adhesive bandage may often not be sufficient to close more severe or deeper wounds.

[0057] In the wound healing process, injured tissue is repaired, specialized tissue is regenerated, and new tissue is reorganized. Three major phases are an inflammation stage of zero to three days, a proliferation stage of three to twelve days, and a remodeling phase of a few days to six months or more. In the inflammation phase, platelet aggregation and clotting form a matrix which traps plasma proteins and blood cells and induces the in-migration of various cells from surrounding tissues. In the cellular proliferation phase, new connective or granulation tissue and blood vessels form. In the remodeling phase, granulation tissue is replaced by a network of collagen and elastin fibers producing scar tissue. Each of these phases is at least partly dependent on signaling by growth factors produced by healing and adjacent tissue. The inventive bandages and methods may enhance wound healing during any of these phases. We have found that healing of wounds may be improved when wound dressings include compositions that contain cell culture medium that has been conditioned by growth of human stem cells. These dressings supply exogenous growth factors derived from expansion of the stem cells in tissue culture. These exogenous growth factors can augment the response generated by the wound healing process itself.

Stem cell culture medium

[0058] As discussed above, growth of such adipose-derived stem cells includes supply of nutrients for the cells through provision of an aqueous culture medium. Cells grown in culture deliver to the medium stem cell products including growth factors and cytokines as well as metabolic products. Culture medium removed after exposure to cells includes stem cell products as well as residual components of the original medium, such as essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, and nucleosides. Cell culture medium attempts to supply the components necessary to meet the nutritional needs required to grow cells in a controlled, artificial and in vitro environment. Nutrient formulations, pH, and osmolarity may vary depending on the type of cell cultured, on cell density, and on the culture system employed. The scientific literature includes description of many cell culture medium formulations; a number of such media are commercially available. Conditioned medium also contains a variety of cellular metabolites and secreted proteins, including, for example, biologically active growth factors, inflammatory mediators and other extracellular proteins. Examples of suitable culture media are Dulbecco's Modified Eagle's Medium and RPMI 1640. Such media may be supplemented by other nutrients, growth supporting materials, or antibiotics as is known in the art. An exemplary process of producing a human adipose derived stem cell conditioned ("HADSCC") medium is that described in PCT US2014/034738, commonly assigned with this application, the specification of which is incorporated by reference.

[0059] In some embodiments the bandages of the invention include a HADSCC medium where the media is supplemented with nanosilver particulates before exposure to the human adipose-derived stem cell culture. In these embodiments, the stem cells are cultured in the presence of the nanosilver particulates. Nanosilver can affect the healing of damaged tissue when applied during wound healing or burn healing. Nanosilver may also affect the growth of human adipose-derived stem cells in culture and their production of stem cell products including growth factors, cytokines, stress proteins, and nutrients. Thus HADSCC medium where the stem cells are cultured in the presence of the nanosilver particulates ("HADSCC silver medium") may contain a different mix of stem cell products than HADSCC mediu m where the stem cells are cultured in the absence of the nanosilver particulates. In some embodiments where the media is supplemented with nanosilver particulates before exposure to the human adipose- derived stem cell culture cells, the media may be otherwise free of or have reduced amounts of antimicrobials such as antibiotics. HADSCC silver medium (other than the presence of nanosilver and possible absence of antibiotics) may be prepared by a similar process to that of HADSCC media without nanosilver. In other embodiments, media harvested from stem cells cultured without nanosilver may be su pplemented with nanosilver after harvesting the conditioned media. [0060] In some embodiments HADSCC medium (or HADSCC silver medium) may be further prepared by concentration or by nanoencapsulation or both. Concentration preparation steps reduce the amount of water in the medium and consequently concentrate the active components of the medium. This step may also serve to desalt the material by passing small ions. Concentration may be performed by any of a variety of methods known in the art, including centrifugal filtration, dialysis, tangential flow filtration, or exposure to water absorbing polymers. In some embodiments, HADSCC medium may be concentrated by centrifugal filtration using a Centricon^® or Microcon^® centrifugal filter devices according to the directions supplied with device by its manufacturer, EMD Millipore of Billerica, Massachusetts. Centricon^® and Microcon^® are registered trademarks of EMD Millipore. Using such a device, active components of the HADSCC medium may concentrated by from about five to about 200 times. We have found particular efficacy for subsequent processing when the active components of the HADSCC medium are concentrated about 50 times. The media treated by this process (or any similar process known in the art) will be referred to as concentrated media, but unless indicated otherwise, subsequent use of the term HADSCC medium includes concentrated media as well as the HADSCC medium as harvested.

HADSCC media from cells cultured without nanosilver may receive nanosilver either before or after concentration.

[0061] HADSCC medium (including concentrated media) may be treated by nanoencapsulation to help enhance stability by protecting the HADSCC medium from exposure to environmental materials or conditions that may degrade the activity of the active materials. Nanoencapsulation may also serve to control the release of active materials to a desired time (e.g. when exposed to a solvent) or at a desired rate. Nanoencapsulation may be performed by any method known in the art, including those reviewed by Subhash Chandra Yadav, Avnesh Kumari, and Ramdhan Yadav in their paper entitled Development of peptide and protein nanotherapeutics by nanoencapsulation and nanobioconjugation published in Peptides 32 pp. 173-187 (2011). This review is hereby incorporated by reference for its disclosure of methods of nanoencapsulation.

[0062] In some embodiments, a suitable method of nanoencapsulation includes emulsification polymerization using aqueous phase methacrylate monomer and a photoinitator such as benzoin ethyl ether emulsified with HADSCC medium with polyethylene oxide as a stabilizer and exposure to UV light after emulsification to produce poly(methacrylate) encapsulated active components of HADSCC medium. The nanocapsules may range from about 50 to about 1000 nm in diameter. While the nanocapsules may be close to monodiserse (depending on the method of preparation), in some embodiments, the size of nanocapsules may be deliberately widely distributed to control the rate of release of active materials. Widely distributed populations of nanocapsules may be prepared by altering the conditions of emulsification during encapsulation or by mixing two or more batches of nanocapsules with different size.

[0063] In other embodiments, nanoencapsulated HADSCC medium may be prepared as phospholipid nano-emulsions or nano-liposomes.

[0064] In other embodiments, nanoencapsulated HADSCC medium may be prepared using the apparatus and method described in US patent publication 2008/0182019 entitled Hollow Microsphere Particle Generator. This publication is hereby incorporated by reference for its disclosure of methods of encapsulation of aqueous phase materials.

[0065] Nanoencapsules containing HADSCC medium may be washed by dialysis, by centrifugal filtration, by tangential flow filtration, or by centrifugation and decanting, or by other techniques known in the art, to produce washed nanoencapsulated HADSCC medium. Washing helps remove unreacted monomers or initiator as well as materials not incorporated in nanocapsules. Alternatively, and depending on the materials used in the encapsulation process, nanoencapsulated medium may be used without further processing. HADSCC media from cells cultured without nanosilver may receive nanosilver either before or after nanoencapsulation. After washing, nanoencapsulated media may be resuspended in a buffer, in sterile saline, in water, or in a suspension containing other materials. In some embodiments, nanoencapsulated HADSCC media from cells cultured without nanosilver may be resuspended in buffer or solvent containing nanosilver.

[0066] In some embodiments, nanoencapsulated HADSCC medium may be applied directly to wound dressings and dried in place, as by air drying. The media encapsulated as described above (and by similar processes known in the art) will be referred to as nanoencapsulated media, but unless indicated otherwise, subsequent use of the term HADSCC medium (or media) includes nanoencapsulated media. In other embodiments, otherwise untreated HADSCC medium or concentrated HADSCC medium may be applied to wound dressings and dried in place. In some embodiments these materials may be mixed with other materials such as nanosilver (in the case when the HADSCC media was derived from cu Iture without nanosilver), antimicrobials, antioxidants, or support materials such as thickeners that help to retain the materials in place.

Compositions [0067] In other embodiments, HADSCC media (including HADSCC silver media), or

nanoencapsulated media may be mixed with other components prior to application to dressings or bandages. These embodiments of the invention may include support ingredients such as oils, vitamins, and emulsifying agents. Oils may include any of a variety of oils helpful to form an emulsion with the aqueous media components. Exemplary oils include coconut oil, avocado oil, neem oil, rosemary oil, manuka oil, safflower oil, and geranium oil. Emulsifying agents may include commercial cold process waxes such as Jeesperse^® CPW-CG-T (a mixture of cetyl alcohol, sodium acrylate, sodium acryloyl dimethyl taurate copolymer, glyceryl monostearate, and caprylic triglyceride). Jeesperse is a registered trademark of Jeen International Corporation of Fairfield, New Jersey. Other support ingredients may also be present.

[0068] In some embodiments the bandages may include ingredients that make them more suitable for treatment of burns as opposed to other wounds. Bandages more suitable for treatment of burns include vitamin E (Tocopheryl Acetate) in amounts of from about 1-5% by weight of the composition. Bandages more suitable for treatment of wounds may include coconut oil in amounts of from about 1- 5% by weight of the composition.

[0069] We have found that a suitable composition for inclusion in bandages includes about 40% to about 60% by weight of HADSCC media or HADSCC silver media. Oils may range 5-20% with emulsifying agents in the range of 10-20% of the mixture. The balance may be other aqueous ingredients such as aloe vera gel and other plant extracts.

[0070] Without intent to be bound by theory, Inventors believe that the beneficial effects of combining nanosilver particles and HADSSC media may arise because the mix of stem cell products from HADSSC media or from HADSSC silver media complements the nanosilver-activated wound healing response. HADSSC silver media may include especially complementary stem cell products. Further, nanosilver particulates have in some instances been associated with cytotoxic effects. These negative effects of nanosilver may be at least in part ameliorated by the growth factors contained in the HADSSC medium.

Dressings

[0071] Dressings may take many forms, but a dressing is generally a flexible substrate, roughly planar, that conforms to the portion of the body including the wound and protects the wound from the environment. Dressings may hold the edges of wound together, absorb and remove fluids from a wound, add moisture when required, or help to contain and apply medications to the wound. Dressings are generally sterilized to avoid introducing infectious organisms. Any materials added to dressing must be capable of withstanding a sterilization procedure. Sterile dressings suitable for use in embodiments of the invention include textiles, polymers, or composites. Textiles may be woven, such as traditional gauze, or nonwoven sheet or web structures bonded together by entangling fiber or filaments. In other embodiments, the sterile dressing may be a polymer film, foam, semi-solid gel, or pad. Suitable composite materials may include multi-layer assemblages of absorbent, bibulous, or hydrophilic materials covered by protective layers that may either pass or prevent passage of moisture. In some embodiments, the wound-facing surface may include a polymer gel that prevents adherence to the wound.

[0072] The bandage may also include a substance or element for fixing of the bandage to a wound, such as a tie, an adhesive, a tape, a compression stocking, or an adherent gel. The sterile dressing may have a wound-facing surface with a peripheral area and a central area. For example, the protective layers may extend beyond the boundary of the absorbent, bibulous, or hydrophilic materials or of a nonadherent wound-facing surface in a peripheral region that includes adhesives to hold the bandage in position. The emollient base containing the HADSCC medium may be applied to the central area.

[0073] In other embodiments, the sterile dressing may include a wound-facing surface that includes an adhesive over substantially the entirety of the wound-facing surface. The sterile dressing includes a second surface opposing the wound-facing surface, and the emollient base may applied to the second su rface. This embodiment may be used primarily to bring together edges of a wound, so adhesive may be required over the entire surface.

[0074] The invention also includes a method including the steps of applying to a wound a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium is dispersed in an emollient base and the emollient base is applied to the sterile dressing. The bandage is then secured to the wound. In some embodiments, the method also includes adding a liquid to the bandage before applying the bandage to the wound. In some embodiments the composition is dried and the liquid rehyd rates the dried composition. In other embodiments, the emollient base substantially immobilizes the conditioned medium and the liquid elutes the conditioned medium from the bandage. The wound may be any of a dermal wound, an epidermal wound, a burn, an infectious lesion, a surgical site, an ulcer, or a scar.

Electroactive dressings

[0075] The invention also includes electroactive dressings treated with a conditioned medium or with cells. The electroactive dressings function, when wetted by a conductive fluid such as the treatment gels of the invention, as electrical batteries that cause a flow of current between the different electrodes. The current is not limited to the dressing but flows in a response to electrical fields determined by the geometry of the electrodes and of the dressing as applied to the skin. The current flows are generally below the level of human perception as they are limited in magnitude to the order of microamperes. These currents, and the voltages that produce them, aid healing by, among other possible mechanisms, encouraging the migration of cells into the treated region. The currents may include currents of ions, such as silver ions, that have antimicrobial or wound treatment properties.

[0076] In some embodiments, HADSCC medium may be dried onto an electroactive dressing. A preferred drying process includes adding the medium to the dressing while the medium is at low temperature, preferably about 4° Celsius. The dressing may then be rapidly frozen and then dried so as to prevent extended oxidation and reduction of the electrodes. In use, the dressing with dried HADSCC medium may be applied to the skin with a conventional conductive gel composition.

[0077] The HADSCC medium may be nanoencapsulated as described for other embodiments. The conditioned medium may be dispersed in an emollient base and the emollient base applied to the sterile electroactive dressing. The emollient base containing the nanoencapsulated HADSCC medium may be dried onto an electroactive dressing as described above.

[0078] The invention includes treatment gel suspensions containing ionic conductive materials in combination with a microcurrent-providing membrane. The currents are produced when electrodes from complementary half cells within a dressing are joined by the gel into a battery; electrical fields from these batteries extend into tissue under treatment. The gel may include an HADSCC medium. In other embodiments, the gels may include living adipose-derived stem cells and a support medium.

[0079] Complementary electrical half-cells are half cells that include at least two electrodes with different electrochemical potentials under operable conditions. Operable conditions means when the half cells are co-wetted by an ionically conductive fluid such as the described treatment gels of the invention.

[0080] Fig. 1 illustrates a prior art electroactive dressing useful in some embodiments of the invention. Dressing 10 includes a flexible sheet having front surface 14 and rear surface 19 parallel to front surface 14. Dressing 10 is thin with respect to its linear extent. Its thickness may be about 0.030 inches. Dressing 10 may be bibulous and porous to promote wetting by the treatment gel and to retain electrode materials. Front surface 14 includes two types of electrodes formed by deposition of metal. The metal may be deposited as particulate suspensions by conventional printing processes. The particulates may be entrapped within the interstices of the porous body or may be attached to the su rface of the body through the use of binding agents within the suspensions.

[0081] In embodiments, the invention includes a dressing that may be similar to that of Fig. 1. Multiple first electrodes 18 form a regular array of spots interspersed or intercalated with multiple second electrode spots 16. While the illustrated embodiment shows electrodes 18 of different size from electrodes 16, this arrangement depends on the relative surface area of the individual particulates. In other embodiments, the spots may of similar sizes or the spots may include more complex geometry such as the small void visible in the center of each first electrode 18. The spacing of the spots may be about 0.5 to about 5 mm.

[0082] Fig. 2 illustrates a second electroactive dressing useful in some embodiments of the invention. Dressing 20 is similar to dressing 10 except that the distribution of metallic electrodes differs from that of dressing 10. Dressing 20 includes a flexible sheet having front surface 28 and rear surface 29 parallel to front surface 28. The flexible sheet of dressing 20 may be hydroph ilic to help retain aqueous liquids in close proximity to the metallic electrodes. Suitable materials include cellulosic materials such as gauze fabric or paper, glass fiber materials, or hydrophilic polymers such as acrylics, nylons, other polymers treated to provide a hydrophilic surface, or a combination of any of these materials.

[0083] Multiple first electrodes 22 may be deposited in a regular array of spots. In some embodiments, first electrodes 22 may be deposited in a rectangular grid. Second electrodes 24 are deposited as a rectangular grid of circles, with each second electrode 24 surrounding one of the first electrodes 22. Additional first electrodes are deposited as spots 26 in a second rectangular grid offset from the grid of first electrodes 22. Dressing 22, when wetted by a conductive treatment gel, may produce a different pattern of voltages and currents that may be more appropriate for some skin conditions than dressing 10. The choice of dressing may depend in part on the desired depth of the treatment area of the affected skin. Without intent to be bound by theory, Inventors believe that the structure of dressing 20 causes a deeper penetration of electric fields into tissue underlying dressing 20 than does dressing 10.

[0084] The currents produced by suitably applied dressings may decrease over time as the available material of the electrodes is either oxidized or reduced. In some embodiments, the integrated current (over the application period of the dressing) may be controlled by adjusting the size of one or both of the electrodes. A smaller electrode is more rapidly depleted, so that a dressing including smaller electrodes delivers less current over the life of the dressing. On the other hand, a smaller electrode permits more precise targeting of the electric field because of the dominance of fringing field effects. In embodiments, the individual electrodes may be between about 0.1 mm and 10 mm in diameter. In some embodiments, electrode feature size (diameters of spots and width of lines or rings) may be about 0.5 mm.

[0085] The intensity and duration of the current may also be adjusted to some extent by the depth of deposition of the electrode materials. This in turn depends on both the thickness of the dressing and on the distribution of electrode materials in that depth. A thicker dressing can hold more electrode materials, but some of the materials are thereby more distant from the treated site, decreasing the effective penetration depth of the electric field. Further, thick materials are generally less flexible and more difficult to apply comfortably to a treated area, though they may have greater mechanical durability for longer term use. In some embodiments, dressing thickness may be about 0.003 to about 0.050 inches. Short term dressings (generally intended for one day or less of application) may have thicknesses of about 0.003 to about 0.02 inches. Longer term dressings (intended for more than one day of treatment before changing) may have thickness of about 0.02 to about 0.05 inches.

Treatment gels

[0086] The compositions of the invention also include treatment gels that are applied between electroactive dressings and the skin to be treated. These compositions and dressings may need to remain in place for hours or days at a time. Accordingly, the compositions contain thickening or gelling agents that serve to reduce flow and evaporation and retain the compositions in proximity to the application site.

[0087] Appropriate thickening or gelling agents include methylcellulose, hydroxypropyl methylcellulose, and sodium carboxymethyl cellulose, among others. The weight proportions of thickening or gelling agents depends on the particular properties of the materials. Gelling agents may be used concentrations of 0.5% to 10%, depending on the agent. In one embodiment, type 7H3SXF of Aqualon^® CMC brand of sodium carboxymethylcellulose available from Hercules Incorporated of Wilmington, Delaware may be mixed at a weight concentration 1.00%. Aqualon is a registered trademark of Hercules Incorporated.

[0088] In some embodiments the compositions include a treatment gel that has a viscosity of 3000cP. In other embodiments, the viscosity may be higher, ranging between about 5000 and about 20000cP. The choice of viscosity depends on the nature of the dressing, the extent of the skin treatment area, the length of time the dressing is to be applied between changes, and the environmental conditions. In dry or hot conditions where the dressing is expected to remain in place for extended periods (one day or longer), a higher viscosity is advantageous because it reduces liquid loss. For shorter durations and for small areas, a lower viscosity is preferred because it may be more easily and comfortably applied.

[0089] The treatment gel may contain additional ingredients such as antimicrobials, added growth factors, peptides, emollients, oils, or emulsifying agents. Antimicrobials include nanosilver materials or antibiotics and serve to prevent growth of infectious agents or other bacteria. Nanosilver materials may also serve to more directly promote healing. The treatment gel may contain 0.0025 to 0.5% nanosilver particulate by weight of the gel. In embodiments containing culture medium (including HASCC medium) nanosilver may be present in similar concentration by weight of the medium rather than of the treatment gel as a whole. The balance of the treatment gels may include other materials such as those detailed in the Examples.

[0090] A composition used in combination with an electroactive dressing needs to provide a conductive electrical path between the half cells of the dressing and between the dressing and the skin. In some embodiments, the ionic concentration may be approximately that of human interstitial fluid or of human blood, ranging about 50 mEq/L to about 200 mEq/L and in some embodiments about 140 mEq/L sodium chloride equivalent. The compositions may be diluted with deionized water or augmented with sodium chloride or other ionic components to adjust the final concentration to within this range. However, the compositions of the invention contain relatively large concentrations (exceeding 50%) of HADSCC Media and other components having appreciable ionic concentration. We have found that no adjustment is required for some embodiments.

[0091] Silver is an effective killing agent against many types of bacteria, viruses, and fungi. It is widely used as a microbicide, as a preservative, and as a deodorant. Both preservative and deodorant properties are commonly ascribed to its microbicidal activity. Silver may also enhance the antibacterial activity of various antibiotics such as penicillin, erythromycin, and vancomycin. Reduced silver particles with size range below about 100 nm are commonly called nanosilver. Nanosilver particles frequently are capped with surface chemical groups that support their stability and dispersion in aqueous suspension.

[0092] Nanosilver dispersions have anti-inflammatory activity in human application; nanosilver may alter the expression of enzymes that are important in inflammatory and tissue repair processes, such as matrix metallo-proteinases. In addition, nanosilver can modulate cytokines involved in wound healing such as by suppressing expression of interleukin (IL)-12, and IL-1 and of TNF-a, and it may induce apoptosis of inflammatory cells. The magnitude of the anti-inflammatory effects of nanosilver is related to the exposure of the treated tissue to reduced silver atoms and thus on a per gram basis depends on the size of the nanosilver particulates. The enhanced effects from smaller particles may be greater than linear with surface area as particles approach a few hundred atoms due to quantum confinement effects. To some extent the effects also depend on the shape of the particles as different shapes expose different crystalline planes with different packing density of silver atoms. The size producing an optimum combination of properties depends on the particular microenvironment, the tissue treated, and the effect measured.

[0093] In some embodiments, the invention includes a treatment gel composition that includes a conditioned medium and a nanosilver particulate. The conditioned medium may be harvested from culture of human adipocyte-derived stem cells. The stem cells may be cultured in the presence of the nanosilver particulate. The culture medium may include one of Dulbecco's Modified Eagle's Medium or RPMI 1640 medium containing 0.0025 - 0.5% nanosilver particulate by weight of the medium.

[0094] In other embodiments, the stem cells are cultured in medium that does not contain a nanosilver particulate. Instead the conditioned medium is compounded with the nanosilver particulate after the media is harvested from the stem cell culture.

[0095] In any of these embodiments, the nanosilver particulate may form about 0.001 - 0.2% by weight of the composition. The nanosilver particulate includes a stabilizing coating, such as a coating including one or more of citrate, tannic acid, polyvinylpyrrolidone, silica, polyethylene glycol, oligonucleotides, or a peptide. In any of these embodiments, the nanosilver particulate may include silver particles have a size range of about 1-10 nm.

[0096] In some embodiments the treatment gels of the invention include an HADSCC medium where the media is supplemented with nanosilver particulates before exposure to the human adipose- derived stem cell culture. In these embodiments, the stem cells are cultured in the presence of the nanosilver particulates. Just as nanosilver can affect the healing of damaged tissue when applied during wound healing or burn healing, so also may nanosilver affect the growth of human adipose-derived stem cells in culture and their production of stem cell products including growth factors, cytokines, stress proteins, and nutrients. Thus HADSCC medium where the stem cells are cultured in the presence of the nanosilver particulates ("HADSCC silver medium") may contain a different mix of stem cell products than HADSCC medium where the stem cells are cultured in the absence of the nanosilver particulates. In some embodiments where the media is supplemented with nanosilver particulates before exposure to the human adipose-derived stem cell culture cells, the media may be otherwise free of or have reduced amounts of antimicrobials such as antibiotics. HADSCC silver medium (other than the presence of nanosilver and possible absence of antibiotics) may be prepared by a similar process to that of HADSCC media without nanosilver.

[0097] In other embodiments the treatment gels may include a HADSCC medium where the media is compounded with nanosilver particulates after the media is harvested.

[0098] Both embodiments containing HADSCC silver media and those containing HADSCC media compounded with nanosilver particulates after the media is harvested contain nanosilver particulates as well as stem cell products, but the mixture of stem cell products may differ because of the effect of the nanosilver particulates during the growth of the stem cells.

[0099] In some embodiments, the treatment gels include autologous human adipose-derived stem cells supported in a culture medium. The cells may be present at a concentration that ranges from about 103 to about 107 cells per mL and in some embodiments from about 5 x 105 to about 3 x 106 cells per mL. The cells may be cryopreserved but are preferably freshly harvested from culture vessels immediately before use. Gels containing living cells may include added culture medium (without any serum products) to support the cells and antioxidant preservatives such as ascorbic acid. Examples of suitable culture media are Dulbecco's Modified Eagle's Medium and RPMI 1640. Such media may be su pplemented by other nutrients, growth supporting materials, or antibiotics as is known in the art. However such culture media do not contain any serum products, including any bovine fetal calf serum.

[00100] The invention includes a method of treating a skin condition in a human. The steps of the method 100 are illustrated schematically in Fig. 3. At step 102, a treatment gel an electroactive dressing as described above provided. At step 104, a user applies the treatment gel either to the surface of the skin to be treated or to the surface of the dressing containing the electrodes. At step 106, the user applies the dressing to the skin. The dressing may be fastened by any of a number of methods known in the art such as by using an adhesive tape or by a overwrapping bandage. The skin condition treated may be any of a broad variety of conditions including wounds, scars, blemishes, acne, stretch marks, or wrinkles.

[00101] In the prospective Examples below, the listed ingredients (with weighed quantities adjusted for batch size) are typically combined in a cleaned and sterilized tank with moderate mixing or in smaller batches in sterile disposable labware. The ingredients may be added in the order listed, one at a time, mixing well between additions. The first two examples are formulations for conductive treatment gels to be applied between treated skin and electroactive dressings as disclosed above. The third example describes a process of producing nanoencapsulated HADSCC medium and applying it to dressings. The final two examples describe emollient treatment compositions for application to dressings as disclosed above.

Example 1 Conductive Treatment Gel

Ingredient (pH range =6.00-6.80) Wt. Limit (%)

HADSCC Medium 50-70%

Palmitoyl tripeptide-5 /Glycerin 5-10.0%

Acetyl Hexapeptide-8 (Argireline) 5-10.0%

Polysosbate-20 5-10.0%

Acetyl Tetrapeptide-5 (Eyeseryl) 5-10.0%

Aloe Vera Gel 5-10.0%

Pentapeptide-18/Caprylyl Glycol (Leuphasyl) 3-7.0%

Algae Extract/Pullulan 1-5.0%

Grapefruit Seed Extract 1-4.0%

Sodium Carboxymethyl Cellulose 1-3.0%

Phenoxyethanol/Sorbic acid/Caprylyl Glycol 1-1.5%

Sodium Hyaluronate Solution 1-3.0%

Fragrance 1-3.0%

DL-Panthenol (Vitamin B5) 1-3.0%

Niacinamide (Vitamin B3) 1-3.0 %

Green Tea Extract 1-3.0%

Nanosome Copper Peptides 0.001-0.01%

EGF-l(Human Oligopeptide-1) 0.001-0.01%

Example 2: Silver Conductive Treatment Gel

Ingredient Wt. Limit (%) HADSCC Media containing 0.05% nanosilver 70.00%

Palmitoyl tripeptide-5 /Glycerin 5.00%

Acetyl Hexapeptide-8 (Argireline) 5.00%

Polysosbate-20 5.00%

Acetyl Tetrapeptide-5 3.00%

Aloe Vera Gel 2.00%

Pentapeptide-18/Caprylyl Glycol (Leuphasyl) 2.90%

Algae Extract/Pullulan 1.00%

Grapefruit Seed Extract 1.00%

Sodium Hydroxymethlyl Cellulose 1.10%

Phenoxyethanol/Sorbic acid/Caprylyl Glycol 1.00%

Example 3 Nanoencapsulated media applied to bandages

[00102] In some embodiments, nanoencapsulated medium may be applied directly to wound dressings and dried in place, as by air or vacuum-assisted drying. The application process (as well as all processing steps for the HADSCC medium and the subsequent encapsulation and any wash steps take place under sterile conditions (as in a laminar hood or sterile clean room). In some embodiments, component materials or the finished product or packaging may be subject to sterilization steps. Such steps are well known in the art and will not be further discussed.

[00103] Nanoencapsulated media are prepared by harvesting HADSCC medium, concentrating the harvested medium by centrifugal filtration, and nanoencapsulating the concentrated medium. The nanoencapsulated media is subsequently washed, with the nanocapsules resuspended in an application buffer.

[00104] Load 17.5 milliliters of freshly-harvested HADSCC medium to a Centricon^® centrifugal filter with Ultracel PL-30 Membrane and spin at 3,500 x g for 20 minutes to produce 0.35 mL of concentrated medium. The concentrated medium is nanoencapsulated by emulsification polymerization. Emulsify the HADSCC medium with twice the volume of 10% aqueous phase methacrylate monomer and 0.5% benzoin ethyl ether as a photoinitator. Expose to UV light after emulsification to produce poly(methacrylate) nanoencapsulated media. Wash the nanoencapsulated media three times by centrifugation at 3500 x g and aspiration of supernatant. Resuspend the washed nanoencapsulated media to a final volume of 1 mL in sterile normal saline to form an application solution.

[00105] Apply the application solution at a coverage of 0.1 mL per cm2 of sterile cotton gauze and air dry for 24 hours.

[00106] The treated gauze may be packed as a sterile dressing and applied directly to a wound or may first be moistened with sterile water (about O.lmL per cm2).

Example 4 Composition applied to dressings

[00107] The listed ingredients (with weighed quantities adjusted for batch size) are typically combined in a cleaned and sterilized tank with moderate mixing or in smaller batches in sterile disposable labware. The ingredients may be added in the order listed, one at a time, mixing well between additions. The ingredients may be subject to mechanical emulsification during processing. In some embodiments, the HADSCC media may include nanosilver as discussed above and may include the HADSCC media as a suspension of nanoencapsulated media as described in example 3.

Ingredient % by weight of final

mixture

HADSCC Media 40%

Jeesperse^® CPW-CG-T (Cetyl Alcohol/Sodium 10-15%

Acrylate/Sodium Acryloyl Dimethyl Taurate

Copolymer/Glyceryl Monostearate/Caprylic/

Triglyceride)

Butylene Glycol 5-10%

Niacinamide (Vitamin B3) 5-10%

DL-Pantothenic (Vitamin B-5 ) 1-5%

Tetrahexyldecyl Ascorbate (Vitamin C Oily) 1-4%

Glycolic Acid 70% 5-10%

Zinc Oxide/Triethoxycaprylylsilane/C12- 2-5%

Benzoate/DEA OIeth 3 Phosphate Aloe Vera Gel 1-5%

Avocado Oil 0.1-0.5%

Coconut Oil 1-5%

Manuka Oil 0.1-1.5%

Phenoxyethanol/Sorbic Acid/Caprylyl Glycol 1-3%

Example 5 Composition applied to dressings for burns

[00108] The listed ingredients (with weighed quantities adjusted for batch size) are typically combined in a cleaned and sterilized tank with moderate mixing or in smaller batches in sterile disposable labware. The ingredients may be added in the order listed, one at a time, mixing well between additions. The ingredients may be subject to mechanical emulsification during processing. I some embodiments, the HADSCC media may include nanosilver as discussed above a nd may include HADSCC media as a suspension of nanoencapsulated media as described in example 3.

Ingredient % by weight of final

mixture

HADSCC Media 40%

Jeesperse^® CPW-CG-T (Cetyl Alcohol/Sodium 10-15%

Acrylate/Sodium Acryloyl Dimethyl Taurate

Copolymer/Glyceryl Monostearate/Caprylic/

Triglyceride)

Butylene Glycol 5-10%

Niacinamide (Vitamin B3) 5-10%

DL-Pantothenic (Vitamin B-5 ) 1-5%

Tocopheryl Acetate (Vitamin E) 1-5%

Tetrahexyldecyl Ascorbate (Vitamin C Oily) 1-4%

Glycolic Acid 70% 5-10%

Zinc Oxide/Triethoxycaprylylsilane/C12-15 Alkyl 2-5% Benzoate/DEA OIeth 3 Phosphate

Avocado Oil 0.1-0.5%

Manuka Oil 0.1-1.5%

Phenoxyethanol/Sorbic Acid/Caprylyl Glycol 1-3%

Safflower Oil 1-5%

Geranium Oil 0.1-0.5%

[00109] This specification discloses various aspects of the invention with reference to particular embodiments, but it should be understood that any of the features, functions, materials, or

characteristics may be combined with any other of the described features, functions, materials, or characteristics. The description of particular features, functions, materials, or characteristics in connection with a particular embodiment is exemplary only; it should be understood that it is within the knowledge of one skilled in the art to include such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. We intend the scope of the appended claims to encompass such alternative embodiments. Variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this specification and claims include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

[00110] Unless otherwise indicated, all numbers used in the specification and claims are to be understood as being modified in all instances by the term "about." Unless indicated to the contrary, the numerical values in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

[00111] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are intended to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 invention and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non - claimed element essential to the practice of the invention.

Claims

CLAIMS We claim:

1. A skin damage treatment device comprising:

a dressing including a cathode and an anode, each forming an electrical half-cell; and a conductive treatment gel including a human adipose-derived stem cell culture

("HADSCC") medium and a gelling agent,

wherein the treatment gel has a viscosity of at least 3000 cP and ionic salts in the range of about 50 mEq/L to about 200 mEq/L.

2. The device of claim 1, wherein the dressing further includes a flat flexible substrate having a first surface and a second surface parallel to the first surface, the first surface supporting the cathode and the anode, and wherein the cathode and the anode include a first electrode and a second electrode, the second electrode forming a closed ring surrounding the first electrode.

3. The device of claims 1 or 2, wherein the anode includes a plurality of discrete anode spots, and the cathode includes a plurality of discrete cathode spots, the plurality of anode spots intercalated among the plurality of cathode spots.

4. The device of claim 3, wherein treatment gel includes at least 50% by weight of the HADSCC medium and at least about 0.5% by weight of a gelling agent.

5. The device of claim 4, wherein the treatment gel includes at least 1.0% by weight of the gelling agent, and wherein the gelling agent includes a hydroxy methyl cellulose or a carboxymethyl cellulose.

6. A conductive treatment gel comprising:

a gelling agent of at least 0.5% by weight;

a human adipose-derived stem cell;

a culture medium; and

ionic salts at a concentration of about 50 mEq/L to about 200 mEq/L.

7. The conductive treatment gel of claim 6, wherein the gelling agent includes a hydroxymethlyl cellulose or a carboxymethyl cellulose, wherein the gel has a viscosity of at least 3000 cP, and wherein the culture medium includes about 0.0025 to about 0.5% nanosilver particulate by weight of the culture medium.

8. A method of treating a skin condition in a human comprising the steps of: providing a treatment gel and a dressing including complementary electrical half- cells;

applying the treatment gel to the skin or to the dressing; and

applying the dressing to the skin,

wherein the gel contains one of a human adipose-derived stem cell conditioned medium or a human adipose-derived stem cell.

9. The method of claim 8, wherein the gel contains human adipose-derived stem cells autologous to the human, a culture medium, and about 0.0025 to about 0.5% nanosilver particulate by weight of the medium.

10. A bandage comprising:

a sterile dressing; and

a composition including a conditioned medium applied to the sterile dressing, wherein the conditioned medium in contained in a plurality of nanocapsules.

11. The bandage of claim 10, wherein the nanocapsules are applied to the sterile dressing as an aqueous suspension and air dried.

12. The bandage of claim 10 or 11, wherein the aqueous suspension includes a nanosilver particulate.

13. The bandage of claim 12, wherein the conditioned medium is dispersed in an emollient base, and the emollient base is applied to the sterile dressing.

14. The bandage of claim 11, wherein the emollient base includes an oil and an emulsifying agent, wherein the oil forms about 5-20% by weight of the composition, and wherein the emulsifying agent forms about 10-20% of the composition.

15. The bandage of claim 14, wherein the conditioned medium is harvested from culture of human adipocyte-derived stem cells, and wherein the conditioned medium forms about 40% by weight of the composition..

16. A method of treating a wound comprising:

applying the bandage of claim 10 to the wound; and

securing the bandage to the wound.

17. The method of claim 16, wherein the composition is dried and the method further comprises adding a liquid to the bandage to rehydrate the composition.

18. A bandage comprising:

a sterile dressing; and

a composition consisting essentially of:

about 5-30% by weight of an oil;

about 10-30% by weight of an emulsifying agent; and

about 40-60% by weight of a conditioned medium harvested from culture of human adipocyte-derived stem cells.

19. A bandage comprising:

a sterile dressing; and

a conditioned medium applied to the sterile dressing,

wherein the conditioned medium is concentrated from media harvested from culture of human adipose derived stem cells, and wherein conditioned media includes a nanosilver particulate.

20. The bandage of any of claims 10, 18 or 19, wherein the sterile dressing includes a flat flexible su bstrate having a first surface and a second surface parallel to the first surface, the first surface su pporting a cathode and an anode, wherein the cathode and the anode include a first electrode and a second electrode, the second electrode forming a closed ring surrounding the first electrode.