Keratin Based Hydrogel Sheets Prepared from Fabric for Biomedical and Other Applications and Method of Production

Background of the Invention

[0001] The chemical treatment of raw animal products containing keratin (hair, wool, fur skin, horn, hooves, or bird feet, beaks, and feathers) has been shown by the present applicant to produce keratin protein products with reduced, reactive sulfide moieties and/or containing sulfonic acid groups that increase the hydration and solubility of the keratin proteins, resulting in useful products in various fields including, but not limited to, medical, veterinary, and dental applications, orthopedics, wound and pain care, soft tissue bulking and replacement, tissue engineering, implants and coatings, and agricultural uses. Active and useful products have been produced as powders, gels, sheets, both woven and unwoven, hydrogels and solids. The current disclosure extends the known technology to incorporate numerous keratin-containing fabrics and other structures and improves the present technology by allowing some or all of these original structures to be retained and by allowing materials other than keratin (i.e. from fabric blends) to be intimately incorporated into the keratin matrix. [0002] The products of the current disclosure are preferably not derived from raw animal starting products and are potentially safer for medical uses. Processes using raw material as the starting substrate require that the keratin protein materials be further processed in most cases, (for example, cast as a film) and/or incorporated into other forms (for example, a bandage). In the current disclosure the material requires much less, if any, further processing to produce a final product. The current disclosure allows the original structure of the fabric material to be wholly or partially retained, thus allowing the properties of the final product to be determined by the choice of the type of fabric or other structural material used as the starting material1 as well as the processing conditions. Finally, the products and processes of the present disclosure are amenable to continuous processing like that used in the textile industry. Summary [0003] The present disclosure provides in part, for the direct preparation of hydratable sheets, hydrogel sheets, and/or viscoelastic hydrogel sheets by utilizing readily available fabrics and allows the properties or texture of the final product to be determined by the choice of fabric starting material as well as by the processing conditions. The present disclosure utilizes blends of keratin-containing fabric with other natural and synthetic blends to prepare composite sheet materials with natural and synthetic fiber imbedded in an oxidized or reduced keratin matrix. The disclosed materials are also produced from three-dimensional weavings of keratin-based materials allowing the preparation of bulk materials, composite bulk materials, and more complex shapes. The disclosed methods can be used to directly prepare finished products or can be used to prepare intermediate materials to finished products, and are amenable to continuous processing as well as batch processing. [0004] The present disclosure may be described therefore in certain embodiments as a composition that includes a wool containing fabric, wherein the wool, or the fabric has been treated to produce a pre-hydrogel such that the wool in the fabric forms a hydrogel upon contact with water. In preferred embodiments the wool is treated by oxidation and ion exchange. It is understood that the fabric may be treated to partially block a portion of the cystine side chains from oxidation prior to an oxidation step. Such treatment may include any of various methods known in the art, including by not limited to disulfide bonds to thiol containing chemicals such as thioglycolate or β-mercaptoethanol, lanthionization or sulfitolysis. After such pre-treatment, only a portion of the cystine side chains are available for oxidation and the control of the amount of oxidation allows the control of the hydratability characteristics of the hydrogel. Partial oxidation can also be achieved by controlling the oxidation conditions without pretreatment of the fabric. [0005] Any appropriate oxidant may be used, including but not limited to hydrogen peroxides, organic peracids, peroxy carbonates, ammonium sulfate peroxide, benzoyl peroxide or perborates, and in certain preferred embodiments oxidation treatment comprises soaking the fabric in an aqueous solution of from about 1-3% hydrogen peroxide at a temperature of from about 70° to about 100°C for a period of about 1-2 hours. Preferred ion exchange treatment may comprise soaking the fabric in 0.1M NaOH or 0.1 M NH4OH for about 16-20 hours. Preferred lanthionization conditions include soaking the fabric in a solution of a hydroxide base in a non-aqueous solvent at a temperature of 20°-30°C for about 18-24 hours, and in preferred embodiments the fabric is soaked in a non-aqueous solution of 1.5% KOH, NaOH, NH4OH, LiOH, or Ca(OH)2. [0006] hi alternative embodiments the lanthionization treatment may comprise soaking the fabric in a solution of a hydroxide base in an alcohol or aprotic solvent, wherein the solvent may include ethanol, methanol, isopropanol, dimethylformamide, dimethyldisulfoxide, N- methylpyrrolidione, or hexamethylphosphoramide. In yet further embodiments, the lanthionization treatment comprises soaking the fabric in a 1.5% KOH/ETOH solution for a period of about 16-24 hours. [0007] Any type of keratin containing fabric can be used in practicing the present disclosure, and in certain embodiments the preferred fabric is a woven fabric comprising wool or a non-woven fabric comprising wool, and wherein the fabric may be wool felt, flannel, gabardine, worsted wool flannel, worsted wool flannel-cotton blend, worsted wool gabardine, or wool jersey knit. The wool fabric may also be wool blended with cotton, polyester, rayon, silk, or nylon. [0008] In certain embodiments of the disclosure, any of the treated wool containing fabrics disclosed or suggested herein may be used as a wound dressing. The present disclosure thus includes wound dressings made from any of the disclosed treated fabrics. The wound dressing may be in the form of a sheet and is preferably in essentially the same form as the fabric prior to treatment. The treated fabric may be stored in sterile, hermetically sealed packages prior to use and hydrated upon opening, or allowed to hydrate by contact with a wound. The wound dressing of the disclosure, are thus individual wound dressing contained in single use packages or they are continuous rolls of material that are cut to the needed size and shape when used. The disclosed wound dressing thus make excellent field dressings for use by the military, by outdoor sportsmen and women or in any other situation in which a first aid kit is employed. [0009] The treated fabric may further include drags or medicines to deliver to the wound including but not limited to analgesics, antibacterials, antibiotics, antifungals, anti- inflammatories, antivirals, cytokines, growth factors, or combinations of these. In preferred embodiments the hydrogel fabric may also deliver water soluble keratin peptides to a wound during use, either peptides that are added to the fabric during production or that naturally occur in the wool and are released upon hydration or contact with a wound. It is understood that the wound dressings may be used on human and animal subjects. [00010] In certain embodiments the hydrogel forming fabrics may be used in the treatment of chronic wounds or in the production of an artificial skin for use in burns or surgical wounds. In certain embodiments, therefore the fabric may include a plasticizer or a backing material such as a surgical mesh as needed for structural strength. [00011] In certain preferred embodiments, a wool or keratin containing fabric may be woven into a three dimensional shape prior to treatment and maintains its shape after oxidation. Such three dimensional materials may be used as implants for bulking of soft tissue or as spinal disc or intra-articular implants, for example. The three dimensional shapes may also include porous regions for ingrowth of skin, muscle, bone or other tissue types as a tissue engineering scaffold. [00012] As described above, the present disclosure also includes films made from the keratin containing fabrics. Such films may include plasticizers such as glycerol, and preferably from 10-40% w/w glycerol. In preferred embodiments the film is produced by subjecting the fabric and plasticizer to a pressure of about 8,000-8,500 pounds at 130° to 150°C. [00013] In certain embodiments, the wound dressings may include a backing material or polymeric binders. Preferred binders include but are not limited to resorbable binders such as polylactic acid polymers, polyglycolic acid polymers, and polylactic acid-polyglycolic acid copolymers, or non-resorbable binders such as ethyl cellulose, cellulose acetate, and polyethylene. [00014] Throughout this disclosure, unless the context dictates otherwise, the word "comprise" or variations such as "comprises" or "comprising," is understood to mean "includes, but is not limited to" such that other elements that are not explicitly mentioned may also be included. Further, unless the context dictates otherwise, use of the term "a" may mean a singular object or element, or it may mean a plurality, or one or more of such objects or elements. Detailed Description [00015] The present disclosure includes hydratable solid sheets derived from any fabric composed wholly or partly of keratin, both woven and non- woven, and including blends with other natural and synthetic fibers such as cotton, nylon, rayon, silk, polyester, etc. In certain embodiments of the invention the fabric is wool fabric such as wool felt, flannel, gabardine, worsted wool flannel, worsted wool flannel-cotton blend, worsted wool gabardine, wool jersey knit, and the like. In certain embodiments the wool fabric is pretreated by certain processes such as lanthionization, reduction, reduction followed by oxidation, oxidation followed by reduction, and in other embodiments the fabric is directly oxidized without pretreatment in aqueous environment using an oxidizing agent such as hydrogen peroxide, H2O2. Upon oxidation at least some portion of the disulfide bonds (S-S) in cystine linkages are oxidized to form sulfonic acid groups. The sulfonic acid and other acid groups in the keratin can be subsequently ion-exchanged (neutralized) using a base such as NH4OH, NaOH, or KOH which results in a hydratable material, hydrogel, or viscoelastic hydrogel upon the application of water. In each case some or all of the original fabric structure is maintained allowing such properties as the degree of hydration, the texture and surface finish, and the strength to be manipulated based upon the properties of the starting fabric. In certain cases where a fabric blend of keratin-containing fabric with a fabric that does not contain keratin, for example, a worsted wool flannel-cotton blend, is utilized, unique properties such as unidirectional elasticity can be achieved. In still other cases where keratin-containing materials produced from three-dimensional weaving are utilized, bulk materials and more complex shapes can be prepared. Description of Preferred Processes

[00016] In one embodiment of the process, samples of worsted wool gabardine (wwg), 3.01 g, were directly oxidized in 50 mL of a 1.68% w/w H2O2 solution containing 1% w/v acetic acid at 750C for 4 hours. At this time the solution containing the wwg was cooled to ambient temperature, isolated by vacuum filtration, and dried in a vacuum oven. The dried samples were treated with excess 0.1M NaOH overnight, then isolated by decantation and vacuum dried. [00017] In another embodiment of the process, samples of wwg, 3.03 g, were directly oxidized in 50 mL of a 3.00 % w/v H2O2 solution containing 1% w/v acetic acid at 100°C for 2 hours. At this time the solution containing the wwg was cooled to ambient temperature isolated by vacuum filtration, and dried in a vacuum oven. The dried samples were treated with excess 0.1M NaOH overnight, then isolated by decantation and vacuum dried. [00018] In yet another embodiment of the process, 5 squares of worsted wool flannel (wwf), 11.92 g, were mixed with 198 mL of 2% H2O2 and heated to reflux (1000C) over a period of approximately 30 minutes. Reflux was continued for 1.5 hours at which time the reaction mixture was cooled to 25°C, isolated by gravity filtration, washed with deionized water, and dried under vacuum. Next, one square was treated with 200 mL of 0.1 M NH4OH for approximately 18 hours, isolated by decantation, washed with 2 x 200 mL of deionized water, and vacuum dried. The dried samples retained the original fabric structure. This sample exhibited hydrogel properties when treated with water. [00019] In another embodiment of the process where the fabric is pretreated before oxidation: 15 squares of worsted wool flannel, 30.60 g, were mixed with 750 mL of 1.5% w/v KOH in ethanol and allowed to stand for approximately 18 hours. Next the fabric squares were isolated by vacuum filtration and washed with 2 x 800 ml. of deionized water, and dried under vacuum. Next the dried pretreated squares, 34.97 g, were mixed with 581 mL of 2% H2O2 and heated to reflux (1000C) over a period of approximately 30 minutes. Reflux was continued for 1.5 hours at which time the reaction mixture was cooled to 25°C, isolated by gravity filtration, washed with deionized water, and dried under vacuum. Next, one square was treated with 200 niL of 0.1 M NH4OH for approximately 18 hours, isolated by decantation, washed with 2 x 200 mL of deionized water, and vacuum dried. The dried samples retained the original fabric structure. This sample exhibited hydrogel properties when treated with water. [00020] In each of the four embodiments of the process described above films were prepared from the fabric samples directly after oxidation (before treatment with NaOH or NH4OH). The fabric samples were treated with glycerol, from about 10% w/w up to about 30 % w/w, as a plasticizer and pressed in a Carver press at 130° to 15O0C. The resulting films exhibited a continuum of strength ranging from hard and brittle to tough and flexible. Treatment of some of these materials with water yields materials reminiscent of skin. [00021] Numerous advantages of the disclosed compositions and processes have been set forth in the foregoing description, as exemplified by the disclosed preferred embodiments. It will be understood by those of skill in the art, however, that changes may be made in details, particularly in matters of reagents, concentrations, and step order, that do not require undue experimentation. All such changes in materials or processes are understood to be equivalent variants of the disclosure and to fall within the spirit and scope of the invention.