Title:  Keratin-based hydrogel for biomedical applications and method of production

United States Patent:  6,159,496

Assignee:  Keraplast Technologies, Ltd. (San Antonio, TX)

Filed:  August 2, 1999

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.

SUMMARY OF THE INVENTION

The present invention includes a hydrogel formed of cross-linked keratin not requiring an added binding agent. The hydrogel is believed to be bound together by reformed disulfide linkages and hydrogen bonds. A preferred use of the hydrogel is as a wound healing agent. Another preferred use is as a tissue engineering cell scaffold for implant applications. Yet another preferred use is as a skin care product. The hydrogel can be formed from a soluble protein fraction derived from hair. Keratin can be obtained from a number of sources including human or animal hair, and finger or toe nails, with one source being hair of the patient or donors.

The hydrogel can be formed by providing clean, washed, rinsed, and dried hair. The hair is partially oxidized with an oxidizing agent such as peracetic acid. The partial oxidation cleaves some disulfide linkages while leaving others intact. The cleaved bonds can form sulfonic acid residues. The partially oxidized hair can be recovered with filtration, rinsed with deionized water, dried under vacuum, and ground to a powder.

The partially oxidized powder can then have some of the remaining intact disulfide linkages cleaved with a reducing agent such as ammonium thioglycollate in ammonium hydroxide by suspending the powder in such a reducing solution. The protein suspension can be heated to about 60^o for about 4 hours and cooled to room temperature. The cleaved disulfide linkages are reduced to form cysteine groups and cysteine-thioglycollate disulfide groups, solubilizing the protein even 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 application of vacuum at ambient or sub-ambient temperatures.

The supernatant, having keratin with sulfonic acid groups, cysteine groups, and cysteine-thioglycollate disulfide groups, is now oxidized to allow formation of disulfide linkages between protein backbones. The sulfonic acid residues remain as hydrophilic sites within the protein. The hydrophilic sites bind water in the hydrogel.

The hydrogel is thus formed of pure keratin, bound together with disulfide linkages and hydrogen bonds. The hydrogel requires no binders. The keratin hydrogel provides a non-antigenic, mitogenic wound healing agent that maintains wound moisture and provides a scaffold for cell growth for tissue engineered implants. Another application for this keratin gel is as a skin care product.

Keratin has been shown to be biocompatible, non-immunogenic, not to inhibit activated T-cells and therefore not interfere with the normal cell mediated immune response, and to be mitogenic for keratinocytes, fibroblasts, and human microvascular endothelial cells. Keratin has also been shown to promote epithelialization in wound healing studies on rats and humans.

Claim 1 of 19 Claims

What is claimed is:

1. A wound healing agent comprising a hydrogel formed of a keratin composition obtained primarily from human hair, wherein said hydrogel is formed primarily by reformed keratin-to-keratin disulfide links and wherein said keratin includes chemically produced hydrophillic groups resulting in an increased number of anionic groups relative to naturally occurring keratin obtained from human hair.

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