MXPA00005252A - Keratin-based hydrogel for biomedical applications and method of production - Google Patents

Abstract

A keratin hydrogel which can be used as a wound dressing and cell scaffolding. The keratin hydrogel is formed from clean, washed hair by partially oxidizing a significant percentage of disulfide linkages to form cysteic acid groups, while some disulfide linkages remain intact. The partially oxidized hair is treated with a reducing agent, thereby reducing most of the remaining disulfide linkages to cysteine-thioglycollate disulfide and cysteine groups. A soluble fraction of hair is collected and oxidized, such that the reduced sulfur groups are allowed to reform disulfide linkages, thereby binding the keratin together. The cysteic acid groups remain, providing hydrophilic sites within the hydrogel. A higher degree of partial oxidation results in a greater abundance of hydrophilic cysteic acid groups in the hydrogel.

Description

HYDROGEL BASED ON QUERATI NA FOR APPLICATION IS BIO MEDICAS AND M ETHOD OF PRODUCTION Cross Reference to Related Requests This application is related to the Request for Patent of E. U.A. with Serial No., presented on the date thereof, entitled "KERATIN-BASED SHEET MATERIAL FOR BIOMEDICAL APPLICATIONS AND METHOD OF PRODUCTION". The present application is also related to the Patent of E. U.A. No. 5, 358, 935, entitled "NONANTIGENIC KERATI NOUS MATERIAL PROTEI", both incorporated herein by reference.

Field of the invention The present invention relates to materials for wound dressings and tissue engineering scaffolding. More specifically, the present invention relates to a cross-linked keratin hydrogel.

BACKGROUND OF THE INVENTION Chronic wounds can be caused by a variety of circumstances, including surgery, prolonged bed rest, and traumatic injuries. Partial depth wounds may include second degree burns, abrasions, and donor sites of skin grafts. The healing of these wounds can be problematic, especially in cases of diabetes mellitus or immune-chronic disorders. Total depth wounds have no remaining skin, and can be the result of trauma, diabetes (for example, leg ulcers), and venous stasis diseases, which can result in total depth ulcers of the lower extremities. Total depth wounds tend to heal very slowly. Appropriate wound care techniques that include the use of wound dressings are extremely important for the successful management of chronic wounds. Chronic wounds affect an estimated four million people a year, resulting in trillion dollar health care costs. "Treatment of Skin Ulcers with Cultivated Epidermal Allografts", T. Phillips, O. Kehinde, and H. Green. J .Am .Acad. Dermatol; V.21, pp.191 -199 (1989). The process of wound healing involves a complex series of biological interactions at the cellular level that can be grouped into three phases: hemostasis and inflammation; granulation tissue formation and reepithelialization; and remodeling. "Cutaneous Tissue Repair: Basic Biological Considerations", R.A. F. Clark, J. Am. Acad. Dermatol; Vol.13, pp.701 -725 (1985). Keratinocytes (epidermal cells that produce and contain keratin) migrate from the edges of the wound to cover the wound. Growth factors such as transforming growth factor β (TGF-β) play a critical role in stimulating the migration process. The migration occurs optimally under the cover of a wet layer. It has been found that keratins are necessary for reepithelialization. Specifically, K5 and K14 keratin types have been found in lower generation epidermal cells, and K1 and K10 types have been found in higher differentiated cells. Wound Healing: Biochemical to Clinical Aspect, I K. Cohen, R. F. Diegleman, and W. J. Lindblad, eds; W.W. Saunders Company, 1992. Keratin types K6 and K1 0 are thought to be present in wounds that are healing, but not in normal skin. Keratins are major structural proteins of all types of epithelial cells and appear to play an important role in wound healing. An optimal bandage for wounds protects injured tissue, keeps the environment humid, is permeable to water, maintains microbial control, provides healing agents to the wound site, and is easy to apply, does not require frequent changes, is non-toxic and not antigenic. Although not ideal for chronic wounds, several wound dressings are currently on the market, including occlusive dressings, nonadherent bandages, absorbent bandages, and bandages in the form of sheets, foams, powders and gels. Wound Management and Dressing, S. Thomas, The Pharmaceutical Press, London. 1990. Attempts have been made to provide improved bandages that can aid in the wound healing process using biological materials as growth factors. To date, it has been proven that these biologicals are very costly and show minimal clinical relevance in accelerating the healing process of chronic wounds. In cases of severe total depth injuries, autografts (skin grafts from the patient's body) are very often used. Although the graft is non-antigenic, it must be cultured from a donor site in the patient's body, creating an additional lesion. In addition, the availability of autologous tissue may not be adequate. Allografts (skin grafts from donors other than the patient) are also used when donor sites are not an option. The allografts essentially provide a "wound dressing" that provides a moist layer permeable to water, but is usually rejected by the patient within two weeks and does not become part of the new epidermis. What would be desirable and has not been provided so far is a wound dressing that protects the injured tissue, maintains a moist environment, is water permeable, easy to apply, does not require frequent changes, is non-toxic and non-antigenic, and Most importantly, provide effective healing agents at the wound site. Tissue engineering is a rapidly growing field that includes a number of technologies aimed at replacing or restoring organ and tissue function. The consistent success of a tissue engineering implant lies in the invention of a biocompatible mitogenic material that can successfully support cell differentiation and growth and integrate with existing tissue.

Such scaffolding material can broadly advance the state of tissue technology and result in a wide range of tissue engineering implants containing cellular components such as osteoblasts, chondrocytes, keratinocytes, and hepatocytes to restore or replace bone, cartilage, skin and liver tissue respectively.

SUMMARY OF THE INVENTION The present invention includes a hydrogel formed from a crosslinked keratin that does not require an added linking agent. It is believed that the hydrogel is bound by the reformed disulfide bonds and the hydrogen bonds. A preferred use of the hydrogel is as a wound healing agent. Another preferred use is as tissue engineering cellular scaffolding for implant applications. Still another preferred use is as a skin care product. The hydrogel can be formed from a fraction of soluble protein derived from the hair. Keratin can be obtained from different sources including human hair or animal hair, and finger and toe nails, with a source being the patient's hair or donors. The hydrogel can be formed by providing clean hair, washing, rinsing and drying. The hair is partially oxidized with an oxidizing agent such as peracetic acid. Partial oxidation penetrates some disulfide bonds while leaving others intact. The penetrated bonds can form sulfonic acid residues. Partially oxidized hair can be recovered with filtration, rinsed with deionized water, vacuum dried, and milled to a powder. The partially oxidized powder may then have some of the remaining intact disulfide bonds penetrated with a reducing agent such as ammonium thioglycolate in ammonium hydroxide by suspending the powder in said reducing solution. The protein suspension can be heated to about 60 ° for about four hours and cooled to room temperature. The penetrated disulfide bonds are reduced to form cysteine groups and cysteine-thioglycolate disulfide groups, solubilizing the protein further. The insoluble keratin fraction is preferably removed from the suspension by centrifuging the suspension and collecting the supernatant. The supernatant is preferably purified using a method such as dialysis. The supernatant can be further concentrated, in one method, by the application of vacuum at ambient or sub-ambient temperatures. The supernatant, which has keratin with sulfonic acid groups, cysteine groups and cysteine-thioglycolate disulfide groups, is oxidized to allow formation of disulfide bonds between the vertebral columns of the proteins. The sulfonic acid residues remain as hydrophilic sites within the protein. The hydrophilic sites bind the water in the hydrogel. In this way, the hydrogel is formed of pure keratin, and binds with the disulfide bonds and hydrogen bonds. The hydrogel does not require links. The keratin hydrogel provides a mitogenic, non-antigenic wound healing agent that maintains wound moisture and provides scaffolding for cell growth for tissue engineering implants. Another application for this keratin gel is as a skin care product. It has been shown that keratin is biocompatible, non-immunogenic, does not inhibit activated T cells and therefore does not interfere with the normal cellular mediated immune response and is mitogenic for keratinocytes, fibroblasts, and human microvascular endothelial cells. It has also been shown that keratin promotes epithelialization in wound healing studies in rats and humans.

Detailed Description of the Preferred Modes In a method according to the present invention, hair is provided, preferably washed and not discolored. The hair is grown from an animal or human resource the patient a human donor is a preferred hair resource, since hair from these remedies is more likely to result in a non-antigenic wound healing product, although animal hair can be acceptable for certain individuals who do not have problems of allergies to animal products. In one method, the hair is washed with Versa-Clean ™ (Fisher Scientific.

Pittsburgh, PA), rinsed with deionized water, and left to air dry. The hair can be oxidized into peracetic acid or another suitable reagent such as H2O2. A preferable treatment uses from 1% to 32% peracetic acid, at a temperature of between 0 ° C and 100 ° C approximately for between 0.5 and 24 hours. One method treats 30 grams of hair with 500 mL of 32% peracetic acid at 4 ° C for 24 hours. It is believed that this treatment with peracetic acid partially oxidizes naturally occurring disulfide bonds to produce a protein with residues of cysteic acid (-CH2S03H), and remaining disulfide bonds. The hair is recovered, preferably with filtration through a coarse vitrified glass, and rinsed several times with deionized water until the rinse solution has a pH of 6.0 or more. The hair can then be dried in a vacuum oven between 20 ° and 50 ° C for 0.5 to 5 days. One method dries the hair in a vacuum oven at 40 ° C for several days. Dry hair can be pulverized and crushed to form a fine powder. A method for grinding hair uses a ceramic grinder and mortar. The keratin powder can be suspended in ammonium thioglycolate. In another method, the powdered keratin powder, derived from hair as described above, is suspended in approximately 3N ammonium hydroxide containing ammonium thioglycolate. Approximately 6 grams of keratin powder can be added per 75 mL of ammonium hydroxide. The potency of the ammonium hydroxide is preferably about 3N and the preferred concentration of ammonium thioglycolate is about 11mL (as thioglycolic acid) per 75mL of ammonium hydroxide the suspension is heated for a sufficient time to solubilize the soluble fraction of the hair. The suspension in one method is heated between 50 ° and 90 ° C from one to 24 hours, followed by cooling. In a preferred method, the suspension is heated to about 60 ° C for about 4 hours and allowed to cool to room temperature. Applicants believe that this treatment penetrates the remaining disulfide bonds to produce cysteine residues in the protein structure. At this point, it is believed that the keratin protein contains cysteic acid, cysteine residues and cysteine-thioglycolate disulfide residues. The proportion of cysteic acid residues and cysteine residues can be controlled by varying the time, temperature, and oxidant concentration in the previously described peracetic acid treatment step. The presence of sulfonic acid residues imparts a hydrophilic property to the hair as well as to the final hydrogel product. After the treatment described above, the keratin fraction resistant to treatment remains, consisting mainly of Beta keratin. This fraction is insoluble to the suspension and removed in a method by centrifugation at approximately 10,000g for approximately ten minutes. The insoluble fraction is set aside for other uses. A thick gelatin type supernatant remains which includes a soluble keratin fraction. The supernatant is collected. The supernatant is preferably purified, using a method such as dialysis. A preferred method uses dialysis against tap water using a dialysis membrane (Spectra / Por ™) having a cut-off of approximately 8000 MW. The resulting solution is preferably concentrated at a concentration of approximately 0.1 grams per mL. The keratin solution is then ready to crosslink to form a hydrogel. In a preferred method, an oxidizing agent is added to the keratin to crosslink the keratin proteins. Preferred oxidizing agents include hydrogen peroxide, organic percents, peroxide carbonates, ammonium sulfate peroxide, benzoyl peroxide, and perborates. The hydrogen peroxide is preferably added to the keratin solution at about 0.5% to about 1.0% v / w, mix well, and let stand at room temperature for several days. A preferred resting time is approximately 3 days. The free flowing solution slowly thickens and becomes a crosslinked hydrogel after about 72 hours. The soluble keratin fraction of the hair is thus partially oxidized to cause the vertebral columns of the proteins to interconnect with the disulfide bonds and make sulfonic acid residues. The partially oxidized keratin is treated with a reducing agent to penetrate some or all of the remaining disulfide bonds, forming thiol groups and thioglycolate disulfide groups and solubilizing more of the keratin proteins. After removing the insoluble fraction, the keratin is oxidized to allow the formation of disulfide lattices. In this way the disulfide lattices are reformed. As used herein, the term "re-forming" refers to broken reticles reformed later, wherein the individual bonds formed later may be, but not necessarily, between the same amino acid pairs of cysteine. As a result, a pure, cross-linked keratin hydrogel is obtained. The hydrogel has sulfonic acid groups that are hydrophilic and bind the water within the hydrogel. The number of sulfonic acid groups corresponds to the degree of oxidation of keratin in the partial oxidation step. Applicants believe that the keratin product made in accordance with this method is suitable for use as a wound healing agent and as mitogenic cell growth scaffold for tissue engineering applications and as a nutrient support for cell growth . It is also suitable for skin care applications. Antibacterial, ointment and biological additives such as growth factors or collagen can be added to the keratin hydrogel.

Experimental Results A keratin-based hydrogel wound healing agent that does not require a binding material was prepared from keratin derived from human hair. Human hair was obtained from men aged 12 to 20 years, washed with Versa-Clean TM (Fisher Scientific Pittsburgh, PA), rinsed with deionized water and allowed to air dry. This hair was subsequently cut into lengths of about 0.63 cm to 5.08 cm, using shears. Thirty grams of this hair were treated with 500 mL of 32% peracetic acid (Aldrich Chemical, Milwaukee, Wl) at 4 ° C for 24 hours. This treatment partially oxidized disulfide bonds. The hair was recovered by filtration through a coarse glass vitrified filter and rinsed several times with deionized water until the rinse solution obtained a pH of 6.0 or more. The hair was dried under vacuum at 40 ° C for several days until it was completely dried and ground to a fine powder with a ceramic grinder and mortar. The resulting material, 19 grams, was used to produce a keratin hydrogel. Six grams of oxidized, powdered hair were suspended in 75 mL of 3N ammonium hydroxide containing 11 mL of ammonium thioglycolate (as thioglycolic acid). The suspension was heated at 60 ° C for 4 hours and then cooled to room temperature. This treatment penetrated the remaining disulfide bonds to produce cysteine residues and cysteine-thioglycolate disulfide residues in the protein structure. An insoluble fraction remained, which was resistant to solubilization by ammonium hydroxide and ammonium thioglycolate. The insoluble fraction, which is thought to be mostly Beta-keratin, was isolated by centrifugation at 10,000 g for 10 minutes. A thick gelatin-type supernatant was removed from the centrifuged material, setting insoluble material aside for use in a related product. The supernatant was subjected to dialysis for 72 hours against tap water using a dialysis membrane with a cut-off of 8000 MW (Spectra / Por TM). The resulting solution was concentrated at 50 mL, under vacuum at a sub-ambient temperature. The solution was treated with 3% hydrogen peroxide added in a proportion of 0.5% to 1.0% v / p, mixed well and allowed to stand at room temperature for 3 days. The solution that freely flowed slowly thickened and became a cross-linked hydrogel after 72 hours. The hydrogel can be used as a wound healing agent or a cellular scaffold. The use of keratin-containing materials to promote wound healing was demonstrated in several experiments. In a first experiment, processed human hair was incubated with a cell culture medium. The medium / hair mixture was passed through a micro filter. Cell lines relevant to wound healing, including human microvascular endothelial cells, keratinocytes and fibroblasts, were placed in a culture using this medium extract. A significant proliferation of these wound healing cells was measured. Keratinocytes proliferated abundantly, fibroblasts proliferated modestly, and endothelial cells proliferated abundantly. The mitogenic activity observed in endothelial cell cultures, fibroblasts, and keratinocytes is further evidence that the keratin protein material is not only biocompatible but also mitogenic with these cell lines. Additional biocompatibility was observed when keratin microfibrils were observed microscopically in direct contact with cells in cell cultures. Specifically, keratinocytes and fibroblasts were observed adhering to and congregating around the microfibrils, indicating that a desirable cellular activity was sustained in this naturally derived biopolymer matrix. In a second experiment, the processed human hair powder was incubated with a cell culture medium. The medium / keratin mixture was passed through a micro filter. This extract of medium was used in proliferation studies with lymphocytes. The lymphocyte cell line did not proliferate, indicating that the material was non-immunogenic. In a third experiment, processed human hair powder was incubated with a cell culture medium. The medium / hair mixture was passed through a micro filter. This extract of medium was used in proliferation studies with activated T-lymphocytes. The T-lymphocytes proliferated normally, indicating that there was no inhibition of the normal cellular immune-mediated response. This showed that there was no inhibition of this important function of immune cells. In a fourth experiment, human hair was chemically treated. This produced a keratin mixture which was then cast to form a sheet and chemically crosslinked to produce a non-soluble keratin sheet. Segments of the leaf were then incubated with human microvascular endothelial cells, keratinocytes and fibroblasts. These cells showed that they could grow and proliferate favorably on the keratin sheet. This indicates that the component cells of the skin proliferate favorably in the presence of the keratin sheet produced by the method described above. In a fifth experiment, twenty hairless rats were injured with dermatome on either side of the dorsal midline, creating a partial depth wound of 0.30 centimeters deep, and 2.0 x 4.0 cm of surface area. Half of the wounds were treated with keratin powder, the other half was not, and both halves were covered with polyurethane bandage. Wounds were observed due to scarring and biopsies were taken on days 0, 2, 4 and 6 for histochemical analysis. Planimetry studies showed 97% of epithelialization of the wounds treated with keratin and 78% of epithelialization of the untreated wounds on day 4. The histological analysis by H &; E revealed microscopically the total epithelialization of the wounds treated with keratin on day 2 and only a partial epithelialization of the untreated wounds on day 2. Histological analyzes on days 4 and 6 also revealed an acceleration of the maturation process of the epithelialization in wounds treated with keratin Human clinical studies are currently being carried out in donor sites for skin grafts. Half of the donor site is treated with sterilized keratin powder and the opposite half is treated in a normal manner, with a non-stick dressing Adaptic TM from Johnson & Johnson. Preliminary results show that the treated halves form epithelial tissue before and mature faster. This was confirmed through both clinical observations and histological results of crushed 4-millimeter biopsies. Subjectively, patients also have much less pain in wounds treated with keratin. Numerous features and advantages of the invention covered by this document have been established in the above description. It should be understood, however, that this description, in many respects, is illustrative only. Changes can be made in details, particularly in matters of configuration, size and order of steps without exceeding the scope of the invention. Of course, the scope of the invention is defined in the language in which the appended claims are expressed.

Claims (32)

1. A keratin hydrogel resulting from the process comprising the steps of: providing hair, wherein the hair is formed of keratin having disulfide bonds; partially oxidizing the hair with a first oxidizing agent, so that a significant percentage of the disulfide bonds in the keratin is converted to cysteic acid groups and a significant percentage of the disulfide bonds remains intact; reducing the hair with a reducing agent, so that a percentage of the remaining disulfide bonds are reduced to cysteine groups and thioglycolate disulfide groups; and oxidizing the hair with a second oxidizing agent, so that a percentage of the reduced cysteine groups and the cysteine-thioglycolate disulfide groups are oxidized to re-form the disulfide bonds, where the reformed disulfide bonds are not necessarily the same existing disulfide bonds provided in the hair; so that the hair is bound by the reformed disulfide bonds and has groups of cysteic acid, wherein the cysteic acid groups are hydrophilic.

2. A keratin hydrogel according to claim 1, wherein between about 1% and 90% of the disulfide bonds are oxidized in the partial oxidation step.

3. A keratin hydrogel according to claim 1, wherein between about 50% to 100% of the remaining disulfide bonds are reduced in the reduction step.

4. A keratin hydrogel according to the claim 1, wherein between about 1% and 100% of the reduced groups are oxidized to re-form the disulfide bonds in the oxidation step.

5. A keratin hydrogel according to claim 1, wherein between about 1% and 90% of the disulfide bonds are oxidized in the partial oxidation step; between about 50% and 100% of the remaining disulfide bonds are reduced in the reduction step; and between about 1% and 100% of the reduced groups is oxidized to re-form the disulfide bonds in the oxidation step.

6. A keratin hydrogel according to claim 1, wherein the first oxidation agents include peracetic acid and the hair is reduced with an alkaline thioglycolate solution.

A keratin hydrogel according to claim 6, wherein the second oxidizing agent is selected from the group consisting of hydrogen peroxide, organic percents, peroxide carbonates, ammonium sulfate peroxide, benzoyl peroxide and perborates.

8. A hydrogel that results from the process comprising the steps of: providing clean and washed hair; oxidize the hair with approximately 32% peracetic acid at about 4 ° C for up to about 24 hours; recover the hair by means of filtration; rinse the hair with deionized rinse water until the rinse water reaches a pH of at least about 6.0; dry the hair under ambient conditions or vacuum at approximately 40 ° C until it is completely dry; crush the hair to obtain a fine powder; suspend the powder in an ammonium hydroxide solution at approximately 3N containing approximately 11 mL of ammonium thioglycolate as thioglycolic acid for each 75 mL of ammonium hydroxide, thereby forming a suspension; heat the suspension to approximately 60 ° C for about 4 hours; cool the suspension to room temperature; centrifuge the suspension at approximately 10,000 g for about 10 minutes and collect the supernatant; purifying the supernatant with dialysis against tap water for about 72 hours using a dialysis membrane having a cut-off of about 8000 MW; concentrate the purified supernatant; oxidize the concentrate by adding approximately 3% hydrogen peroxide at a ratio of 0.5% to 1.0% v / p approximately and mixing well; and let the concentrate stand at room temperature for about 72 hours, thereby forming a cross-linked hydrogel.

9. A process for forming a keratin hydrogel comprising the steps of: providing hair, wherein the hair is formed of keratin having initial disulfide bonds; reacting the hair with a first agent that adds hydrophilic groups to the keratin; reduce the hair with a reducing agent, so that a significant percentage of the disulfide bonds becomes reduced groups; and oxidizing the hair with a second agent, so that a significant percentage of the reduced groups is oxidized to re-form the disulfide bonds, so that the hydrogel is bound by the reformed disulfide bonds and has hydrophilic groups.

A process for forming a keratin hydrogel according to claim 9, wherein the first agent is a first oxidizing agent and the hair is partially oxidized with the first oxidizing agent, so that a significant percentage of the disulfide bonds in the keratin it is oxidized to form the hydrophilic groups and a significant percentage of the disulfide bonds remains un oxidized.

A process for forming a keratin hydrogel according to claim 10, wherein a significant percentage of the disulfide bonds in the keratin is oxidized to cysteic acid groups in the partial oxidation step, and the reduced groups include of cysteine and cysteine-thioglycolate disulfide groups, so that the hydrogel is bound by the reformed disulfide bonds and has groups of cysteic acid, wherein the cysteic acid groups are hydrophilic.

12. A process for forming a keratin hydrogel comprising the steps of: providing vertebrate hair; oxidize hair; suspend the oxidized hair in an ammonium hydroxide solution containing ammonium thioglycolate, thereby forming a suspension; Separate the soluble fraction of the keratin from the suspension; adding an oxidizing agent to the soluble keratin fraction; and let the concentrate sit, with this forming a crosslinked hydrogel.

13. A process for forming a keratin hydrogel according to claim 12, wherein the separation step includes centrifuging the suspension and collecting the supernatant.

14. A process for forming a keratin hydrogel according to claim 13, further comprising purifying the supernatant by means of dialysis.

15. A process for forming a keratin hydrogel according to claim 14, further comprising heating the suspension before centrifuging it.

16. A process for forming a keratin hydrogel according to claim 12, further comprising concentrating the soluble fraction.

17. A process for forming a keratin hydrogel according to claim 12, further comprising concentrating the soluble fraction.

18. A process for forming a keratin hydrogel according to claim 17, wherein the concentrated fraction has a concentration of about 0.1 gram of keratin per ml.

19. A wound healing agent comprising a hydrogel formed from keratin, wherein the keratin protein is linked with covalent disulfide bonds, and the keratin protein have anionic groups responsible for the hydrophilic property.

20. A hydrogel formed from the steps comprising: providing a concentrated keratin solution, wherein the keratin has chemically produced hydrophilic groups and cysteine groups, adding the oxidizing agent to the keratin solution, and allowing the keratin solution to be reticulate, so that at least part of the cysteine groups establish disulfide bonds therebetween thereby forming a crosslinked hydrogel having hydrophilic groups.

21. A hydrogel according to claim 20, wherein the chemically produced hydrophilic groups include cysteine groups.

22. A hydrogel according to claim 21, wherein the keratin includes cysteine-thioglycolate disulfide groups.

23. A hydrogel according to claim 20, wherein the concentrated hydrogel has a concentration in the range of 0.05 to 0.4 grams of keratin per mL.

24. A hydrogel according to claim 22, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide, organic percents, peroxide carbonates, ammonium sulfate peroxides, benzoyl peroxide, and perborates.

25. A hydrogel according to claim 24, wherein the oxidizing agent is hydrogen peroxide.

26. A hydrogel according to claim 20, wherein the keratin is an anionic polyelelyte keratin at a physiological or higher pH.

27. A hydrogel according to claim 21, wherein the gel does not include a binder.

28. A method for treating a wound comprising the steps of: providing a keratin hydrogel, wherein the hydrogel binds with the covalent keratin disulfide bonds; and apply the hydrogel to the wound.

29. A method for treating a wound according to claim 28, wherein the keratin hydrogel is primarily attached with disulfide bonds and includes hydrophilic cysteic acid residues.

30. A keratin hydrogel resulting from the process comprising the steps of: providing keratin, wherein the keratin has initial disulfide bonds; chemically treating the keratin with a first reagent, so that hydrophilic groups are formed in the keratin; reduce keratin with a reducing agent, so that a significant percentage of disulfide bonds are converted to reduced groups; and oxidizing the keratin with an oxidizing agent, so that a significant percentage of the reduced groups are oxidized to re-form the disulfide bonds. so that the hydrogel is bound by the reformed disulfide bonds and has hydrophilic groups.

31. A keratin hydrogel according to claim 30, wherein the first reagent is an oxidizing agent, the hydrophilic groups include cysteic acid formed from the initial disulfide bonds, and part of the initial disulfide bonds remains intact after the step of chemical treatment.

32. A keratin hydrogel according to the claim 31, wherein the reduced groups include cysteine. 00PO607 HYDROGEL BASED ON KERATIN FOR BIOMEDICAL APPLICATIONS AND PRODUCTION METHOD SUMMARY A keratin hydrogel that can be used as a bandage for wounds and as cellular scaffolding. The keratin hydrogel is formed from clean, washed hair by partial oxidation of a significant percentage of disulfide bonds to form cysteic acid groups, while some disulfide bonds remain intact. The partially oxidized hair is treated with a reducing agent, thereby reducing most of the remaining disulfide bonds to cysteine-thioglycolate and cysteine disulfide groups. A soluble fraction of hair is collected and oxidized, in such a way that the reduced sulfur groups are allowed to reform the disulfide bonds, thereby binding the keratin. The cysteic acid groups remain, providing hydrophilic sites inside the hydrogel. A high degree of partial oxidation results in a greater abundance of hydrophilic cysteic acid groups in the hydrogel. * * * * *