U.S. patent application number 10/218100 was filed with the patent office on 2003-02-20 for keratin containing implant material.
This patent application is currently assigned to Southwest Research Institute and Keraplast Technologies, Ltd.. Invention is credited to Blanchard, Cheryl R., Smith, Robert A., Timmons, Scott F..
Application Number | 20030035820 10/218100 |
Document ID | / |
Family ID | 26894356 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035820 |
Kind Code |
A1 |
Timmons, Scott F. ; et
al. |
February 20, 2003 |
Keratin containing implant material
Abstract
Methods for producing thin keratin films, sheets, and bulk
materials, and products formed using these methods. One method
includes providing hair, reducing the hair such that the disulfide
linkages are broken and free cysteine thiol groups formed,
separating out a more soluble keratin fraction in solution, forming
a thin layer from the more soluble fraction, and air drying the
keratin fraction in the presence of oxygen, thereby forming new
disulfide bonds imparting strength to the resulting thin keratin
film. One method includes reducing hair by heating the hair under
nitrogen in an ammonium hydroxide and ammonium thioglycolate
solution followed by centrifuging and collecting the supernatant
containing the more soluble keratin fraction. The more soluble
keratin in this method is precipitated using HCI, removed, and
resuspended in ammonium hydroxide. The keratin solution thus formed
is poured onto a flat surface and allowed to air dry into a thin
keratin film. The film may be used as a wound dressing, a
tissue-engineering scaffold, a diffusion membrane, a coating for
implantable devices, and as a cell encapsulant. In another method,
the keratin solution thus formed is concentrated, poured into a
mold, and allowed to air dry into a three dimensional keratin
product. The resulting bulk product can be used as a cross-linked
implantable biomaterial for soft and hard tissue replacement. In
another method, a keratin solution is emulsified and freeze dried,
forming a porous, open cell keratin material.
Inventors: |
Timmons, Scott F.; (San
Antonio, TX) ; Blanchard, Cheryl R.; (Warsaw, IN)
; Smith, Robert A.; (Jackson, MS) |
Correspondence
Address: |
VINSON & ELKINS, L.L.P.
1001 FANNIN STREET
2300 FIRST CITY TOWER
HOUSTON
TX
77002-6760
US
|
Assignee: |
Southwest Research Institute and
Keraplast Technologies, Ltd.
|
Family ID: |
26894356 |
Appl. No.: |
10/218100 |
Filed: |
August 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10218100 |
Aug 13, 2002 |
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09685315 |
Oct 10, 2000 |
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6432435 |
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09685315 |
Oct 10, 2000 |
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09362244 |
Jul 28, 1999 |
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6159495 |
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09362244 |
Jul 28, 1999 |
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09198998 |
Nov 24, 1998 |
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6110487 |
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09198998 |
Nov 24, 1998 |
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08979526 |
Nov 26, 1997 |
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5948432 |
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Current U.S.
Class: |
424/423 ;
424/93.7 |
Current CPC
Class: |
A61K 35/36 20130101;
C12N 2533/50 20130101; A61L 26/008 20130101; A61K 9/70 20130101;
C07K 14/4741 20130101; A61L 27/60 20130101; C08H 1/06 20130101;
A61L 27/34 20130101; A61K 38/1709 20130101; A61L 15/32 20130101;
A61L 27/227 20130101; A61K 38/39 20130101; C12N 5/0068 20130101;
A61L 26/0047 20130101; A61L 31/047 20130101; Y10S 530/842 20130101;
A61L 31/10 20130101; A61L 27/227 20130101; C08L 89/04 20130101;
A61L 27/34 20130101; C08L 89/04 20130101; A61L 27/34 20130101; C08L
89/00 20130101; A61L 31/047 20130101; C08L 89/04 20130101; A61L
31/10 20130101; C08L 89/00 20130101 |
Class at
Publication: |
424/423 ;
424/93.7 |
International
Class: |
A61K 045/00; A61K
038/17 |
Claims
What is claimed is:
1. A tissue engineering scaffold comprising a keratin having added
hydrophilic groups bound to said keratin, wherein said keratin is
bound together with bonds consisting essentially of
keratin-to-keratin disulfide bonds.
2. The tissue engineering scaffold of claim 1, wherein said keratin
is primarily beta keratin.
3. The tissue engineering scaffold of claim 2, wherein said keratin
is at least 80% beta keratin.
4. The tissue engineering scaffold of claim 1, wherein said keratin
is derived from hair.
5. The tissue engineering scaffold of claim 4, wherein said hair is
human hair
6. The tissue engineering scaffold of claim 1, wherein said keratin
contains sulfonic acid groups.
7. The tissue engineering scaffold of claim 6, wherein said keratin
contains cysteinethioglycollate disulfide residues.
8. The tissue engineering scaffold of claim 2, wherein said keratin
contains sulfonic acid groups.
9. The tissue engineering scaffold of claim 8, wherein said keratin
contains cysteinethioglycollate disulfide residues.
10. A method of engineering tissue comprising the implantation of a
tissue engineering scaffold comprising a keratin having added
hydrophilic groups bound to said keratin, wherein said keratin is
bound together with bonds consisting essentially of
keratin-to-keratin disulfide bonds.
11. The method of claim 10, wherein said keratin is primarily beta
keratin.
12. The method of claim 11, wherein said keratin is at least 80%
beta keratin.
13. The method of claim 10, wherein said keratin is derived from
hair.
14. The method of claim 13, wherein said hair is human hair.
15. The method of claim 10, wherein said keratin contains sulfonic
acid groups.
16. The method of claim 15, wherein said keratin contains
cysteine-thioglycollate disulfide residues.
17. The method of claim 11, wherein said keratin contains sulfonic
acid groups.
18. The method of claim 17, wherein said keratin contains
cysteine-thioglycollate disulfide residues.
19. The method of claim 1, wherein said method if a method for
engineering bone tissue.
20. The method of claim 1, wherein said method if a method for
engineering cartilage tissue.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 09/362,244, filed Jul. 28, 1999, which is a
divisional of U.S. Pat. No. 6,110,487, which is a
continuation-in-part of U.S. Pat. No. 5,948,432.
FIELD OF THE INVENTION
[0002] The present invention is related to products formed from
Keratin derived from hair. More specifically, the present invention
is related to films, sheets, and bulk materials formed from
keratin. The present invention is directed to a cross-linked
keratin based bulk, film or sheet material for use in biomedical
implant, wound dressing, and tissue engineering applications. More
specifically, one aspect of the present invention relates to a
material based primarily on alpha keratin produced by cross-linking
keratin derived from a soluble fraction of keratinous material such
as hair.
BACKGROUND OF THE INVENTION
[0003] Chronic wounds can be caused by a variety of events,
including surgery, prolonged bed rest, and traumatic injuries.
Partial thickness wounds can include second degree bums, abrasions,
and skin graft donor sites. Healing of these wounds can be
problematic, especially in cases of diabetes mellitus or chronic
immune disorders. Full thickness wounds have no skin remaining, and
can be the result of trauma, diabetes (e.g., leg ulcers), and
venous stasis disease, which can cause full thickness ulcers of the
lower extremities. Full thickness wounds tend to heal very slowly.
Proper wound care technique, including the use of wound dressings,
is extremely important to successful chronic wound management.
Chronic wounds affect an estimated four million people a year,
resulting in health care costs in the billions of dollars.
"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).
[0004] The wound healing process involves a complex series of
biological interactions at the cellular level, which 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 manufacture and contain keratin) migrate from
wound edges to cover the wound. Growth factors such as transforming
growth factor-.beta. (TGF-.beta.) play a critical role in
stimulating the migration process. The migration occurs optimally
under the cover of a moist layer. Keratins have been found to be
necessary for reepithelialization. Specifically, keratin types K5
and K14 have been found in the lower, generating, epidermal cells,
and types K1 and K10 have been found in the upper, differentiated
cells. Wound Healing: Biochemical and Clinical Aspects, I. K.
Cohen, R. F. Diegleman, and W. J. Lindblad, eds., W. W. Saunders
Company, 1992. Keratin types K6 and K10 are believed to be present
in healing wounds, but not in normal skin. Keratins are major
structural proteins of all epithelial cell types and appear to play
a major role in wound healing.
[0005] An optimum wound dressing would protect the injured tissue,
maintain a moist environment, be water permeable, maintain
microbial control, deliver healing agents to the wound site, be
easy to apply, not require frequent changes, and be non-toxic and
non-antigenic. Although not ideal for chronic wounds, several wound
dressings are currently on the market, including occlusive
dressings, non-adherent dressings, absorbent dressings, and
dressings in the form of sheets, foams, powders, and gels. Wound
Management and Dressing, S. Thomas, The Pharmaceutical Press,
London, 1990.
[0006] Attempts have been made to provide improved dressings that
would assist in the wound healing process using biological
materials such as growth factors. To date, these biologicals have
proven very costly and have shown minimal clinical relevance in
accelerating the chronic wound healing process. In cases of severe
full thickness wounds, autografts (skin grafts from the patient's
body) are often used. Although the graft is non-antigenic, it must
be harvested from a donor site on the patient's body, creating an
additional wound. In addition, 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.
Allografts essentially provide a "wound dressing" that provides a
moist, water-permeable layer, but is rejected by the patient
usually within two weeks and does not become part of the new
epidermis.
[0007] What would be desirable, and has not heretofore been
provided, is a wound dressing that protects the injured tissue,
maintains a moist environment, is water permeable, is easy to
apply, does not require frequent changes, is non-toxic and
non-antigenic, and most important, delivers effective healing
agents to the wound site.
[0008] Film materials compatible with living tissue are useful for
a number of applications including tissue engineering scaffolding,
diffusion membranes, coatings for implantable devices, and cell
encapsulants. Bulk keratin materials compatible with living tissue
are useful for a number of applications including open cell tissue
engineering scaffolding and bulk, cross-linked biomaterials. Tissue
engineering is a rapidly growing field encompassing a number of
technologies aimed at replacing or restoring tissue and organ
function. The consistent success of a tissue-engineered implant
rests on the invention of a biocompatible, mitogenic material that
can successfully support cell growth and differentiation and
integrate into existing tissue. Such a scaffolding material could
greatly advance the state of the tissue engineering technologies
and result in a wide array of tissue engineered implants containing
cellular components, such as osteoblasts, chondrocytes,
keratinocytes, and hepatocytes, to restore or replace bone,
cartilage, skin, and liver tissue respectively.
[0009] Diffusion membranes are commonly formed of synthetic
polymeric materials, rather than biologically-derived materials.
Diffusion membranes derived from biological materials have the
advantage of enhanced biocompatibility. In particular,
non-antigenic diffusion membranes are compatible with implantation
in the human body and would provide great advantages in controlled
drug release applications.
[0010] Implantable devices, such as pacemakers, stents, orthopedic
implants, urological implants, dental implants, breast implants,
and implants for maxillofacial reconstruction are currently encased
in, or made of, materials including titanium, silicone, stainless
steel, hydroxyapatite, and polyethylene, or encapsulated in
materials such as silicone or polyurethane. These metals, ceramics,
and synthetic polymers have disadvantages related to
biocompatibility and antigenicity which can lead to problems
related to the long term use of these devices. A coating material
derived from biological materials and having non-antigenic and
mitogenic properties would provide a device the advantage of long
term biocompatibility in vivo and potentially extend the useful
lifetime of an implant while decreasing the risk of an allergic or
negative immune response from the host.
[0011] Cell encapsulants such as Chitin/Alginate and bovine-derived
collagen are used to encapsulate mammalian cells for applications
such as tissue engineering/organ regeneration and bacteria for
cloning applications. A non-antigenic, non bioresorbable cell
encapsulant material would have the advantages of providing the
cell with a mitogen and increasing the chances for the cell to
accomplish its tissue engineering function.
[0012] A bulk, cross-linked implantable biomaterial that was
non-antigenic and possessed the appropriate mechanical properties
could be used for maxillofacial restoration, for example, for both
soft and hard tissue replacement. Such a bulk material could also
be used for orthopedic applications as a bone filler and for
cartilage regeneration. A bulk material capable of being implanted
could also be used for neurological applications, such as for nerve
regeneration guides.
[0013] Keratin, often derived from vertebrate hair, has been
processed into various forms. Commonly assigned U.S. Pat. No.
5,358,935 discloses mechanically processing human hair into a
keratinous powder. The hair is bleached, rinsed, dried, chopped,
homogenized, ultrasonicated, and removed from solvent, leaving a
keratin powder. In U.S. Pat. No. 5,047,249, Rothman discusses
activating keratin with a reducing agent and applying the activated
keratin to a wound. Rothman believes the activated keratin thiol
groups will react with thiol groups in the wound tissue and form a
disulfide bond, allowing the keratin to adhere to and protect the
wound.
[0014] Keratin derived materials are believed to be non-antigenic,
particularly when derived from a patient's own keratin. A film
formed from keratin based material would be desirable. A keratin
film able to be used for tissue-engineering scaffolds, diffusion
membranes, implantable device coatings, and cell encapsulants would
be very useful. A solid keratin bulk material would also have great
utility. In addition, a non-antigenic, mitogenic open cell keratin
scaffold would prove highly beneficial for use as a tissue
engineered scaffold to support, nourish, and stimulate cell growth
preceding and following implantation.
SUMMARY OF THE INVENTION
[0015] The present invention includes a sheet formed of
cross-linked keratin not requiring a synthetic binding agent. The
sheet is believed to be bound together by reformed disulfide
linkages and hydrogen bonds. A preferred use of the sheet is as a
wound healing dressing. Another preferred use is as a tissue
engineering cell scaffold for implant applications. The sheet can
be formed from a combination of soluble and insoluble protein
fractions derived from hair, including alpha and beta keratin
fractions. 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 a donor.
[0016] The sheet can be formed by providing an insoluble chemically
modified keratin fraction suspended in water and lowering the pH
until the keratin protein is partially swelled. Partially swelled
is defined as the protein molecule swelling such that the resulting
suspension of keratin particles behaves like a colloidal
suspension. In one method, concentrated sulfuric acid is added
until a pH of less than 1 is reached. Applicants believe the low pH
disrupts the hydrogen bonds which have been rendering the keratin
fraction insoluble, thereby allowing the protein to partially
swell. The partially swelled keratin is then made basic with
ammonium hydroxide. This treatment exchanges the non-volatile acid
with a volatile base, which is removed upon drying. Alternatively,
a volatile acid, such as formic acid, may be employed, eliminating
the requirement for further treatment with a volatile base. The
resulting slurry can then be cast onto a flat surface or mold of
appropriate geometry and surface finish and air dried to produce a
cross-linked keratin sheet. Applicants believe the cross-links
result from the thiol groups re-forming disulfide linkages and from
the amine, and carboxylic acid groups forming hydrogen bonds.
[0017] The resulting sheet is thus formed of pure keratin. 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.
[0018] Another embodiment of the invention includes partially
oxidizing the keratin disulfide linkages to form hydrophilic
groups. One such method includes treating the keratin with peractic
acid to form sulfonic acid groups from a substantial portion, but
not all of, the disulfide bonds. Most of the sulfonic acid groups
remain in the final product as hydrophilic groups, binding water
and hydrating the keratin material. A later reduction step cleaves
many of the remaining disulfide bonds to form cysteine residues.
The partially oxidized and reduced keratin can then be in put in
solution, concentrated, and cast onto a flat surface to oxidize and
re-form disulfide cross-links. In one method, oxygen in air acts as
the oxidizing agent, with the keratin being air dried to form a
film on the flat surface. The moist keratin sheet, consisting
primarily of keratin derived from beta keratin, has the consistency
of moist, thick paper. The sheet dries to a brittle material, which
can be rehydrated to a supple, skin-like material. The rehydrated
sheet has the look and feel of skin while retaining moisture within
the sheet and within the wound. The sheet can be used as a
wound-healing dressing or as a cell-growth scaffold. The sheet can
be cut and shaped as needed before being applied to the wound. The
keratin sheets provide a non-antigenic wound dressing that
maintains wound moisture for migrating epithelial cells and
provides a scaffold for cell growth for tissue engineered implants.
Other applications for this keratin sheet include use as diffusion
membranes and as an encapsulant for cells.
[0019] The present invention includes methods for forming keratin
based thin films, open cell foams, and bulk materials. The thin
films are suitable for use as wound dressings, tissue-engineering
scaffolds, diffusion membranes, coatings for implantable devices,
and cell encapsulants. In one method, cut, washed, rinsed, and
dried vertebrate hair is provided. The hair is reduced with a
reducing agent, such that some of the disulfide linkages are
broken, and a more soluble keratin fraction and a less soluble
keratin fraction formed. The more soluble keratin fraction is
separated, collected, and deposited onto a surface, thereby forming
a layer of the more soluble keratin fraction. The keratin layer is
exposed to an oxidizing agent, such as air, oxygen, or
H.sub.2O.sub.2, and preferably dried. The free thiol groups are
oxidized by the oxidizing agent, the resulting keratin film being
strengthened by the newly formed disulfide bonds. A higher degree
of crosslinking, and therefore strength, can be obtained by the
addition of crosslinking agents such as glutaraldehyde.
[0020] In one method according to the present invention, a keratin
solution is provided, the keratin being dissolved in a first
solvent such aqueous thioglycolate. The keratin has free thiol
groups, produced by methods such as reduction with ammonium
thioglycolate. A second solvent such as hexane or Freon is
provided, the second solvent preferably being substantially
immiscible in the first solvent and the keratin preferably being
substantially insoluble in the second solvent. An emulsion of the
second solvent in the keratin solution can be formed using a
homogenizer. The emulsion is freeze dried, preferably by freezing
the emulsion and removing the first and second solvents under
vacuum, creating a porous keratin material. The porous keratin
material can be warmed to room temperature in the presence of an
oxidizing agent, promoting the formation of disulfide cross-links
between the keratin. In one method, the oxidizing agent is an
oxygen containing gas such as air. In another method, hydrogen
peroxide is mixed with the keratin solution prior to homogenizing.
Applicants believe the resulting material is an open cell scaffold
having substantially spherical voids corresponding to the second
solvent in the emulsion and a cross-linked keratin structure
corresponding to the keratin solution in the emulsion.
[0021] In another method according to the present invention, a
keratin solution is provided, the keratin being dissolved in a
solvent such as aqueous thioglycolate. The keratin has free thiol
groups, produced by methods such as reduction with ammonium
thioglycolate. The keratin can be atomized and sprayed onto a very
cold surface, sufficiently cold to freeze the keratin solution. In
one method, the surface is the surface of a mold. More keratin
solution can be atomized and sprayed over the already frozen
keratin, thereby building up a thicker open cell layer of frozen
keratin. The frozen keratin can be freeze dried by removing at
least a substantial portion of the solvent, and preferably all of
the solvent, under low pressure at low temperature. The keratin
material can be warmed to room temperature in the presence of an
oxidizing agent, promoting the formation of disulfide cross-links
within the keratin solids formed and between the keratin solids
formed. In one method, the oxidizing agent includes gaseous oxygen.
In another method, the oxidizing agent includes hydrogen peroxide
added to the keratin solution. Applicants believe the resulting
structure is an open cell scaffold having substantially spherical
keratin structures corresponding to the atomized keratin and having
voids therebetween. Applicants believe the substantially spherical
keratin structures have disulfide cross-links formed within, and
the structures have disulfide cross-links between structures where
touching each other.
[0022] In one method, according to the invention, hair is cut,
washed, dried, and suspended in ammonium hydroxide containing
ammonium thioglycolate. The suspension is under a nitrogen
atmosphere. The basic ammonium thioglycolate solution serves to
solubilize the keratin and reduce the disulfide cross-links.
Cysteine thiol groups and cysteine thioglycolate groups are formed
from the broken disulfide bonds. The nitrogen atmosphere serves to
prevent oxidation and reformation of disulfide bonds. Heating is
preferably followed by comminuting the hair particles with a tissue
homogenizer followed by further heating under a nitrogen
atmosphere. A fine keratin suspension results.
[0023] The fine keratin suspension is centrifuged, and the
supernatant containing a more soluble keratin fraction is collected
and precipitated out with acid. The precipitate is resuspended in
ammonium hydroxide. The keratin solution is then cast as a thin
film on a surface and allowed to air dry. The air serves to remove
water, concentrate the keratin, and oxidize the cysteine thiol
groups, forming disulfide bridges and strengthening the film.
Further crosslinking can be achieved using chemical means such as
glutaraldehyde. The resulting film is tough and insoluble.
[0024] In one method, cut, washed, rinsed, and dried vertebrate
hair is provided. The hair is reduced with a reducing agent, such
that some of the disulfide linkages are broken, and a more soluble
keratin fraction and a less soluble keratin fraction formed. The
more soluble keratin fraction is separated, collected, and
concentrated, and the more soluble keratin fraction is deposited
into a mold. The concentrated keratin solution in the mold is
exposed to an oxidizing or crosslinking agent and preferably dried.
The free thiol groups are oxidized by the oxidizing agent or
cross-linked by the crosslinking agent, and the keratin
strengthened by newly formed disulfide bonds. In another method,
the concentrated keratin solution is either atomized into a cold
mold or mixed with a polar solvent, emulsified, and freeze-dried to
form an open-cell material. The keratin solution is exposed to an
oxidizing or crosslinking agent, which cross-links and strengthens
the material. A porous keratin material remains.
[0025] In another method according to the present invention, the
more soluble keratin solution is further concentrated, for example,
by air drying or heating under sub-ambient pressure. The
concentrated solution is poured into a mold and allowed to air dry.
The air serves to remove water, concentrate the keratin, and
oxidize the cysteine-thiol groups, forming disulfide bridges and
strengthening the keratin material. The resulting bulk keratin
material is tough and insoluble. In another method, a liquid
oxidizing agent such as hydrogen peroxide is used. In yet another
method, a crosslinking agent such as glutaraldehyde is used.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] Keratin Preprocessing
[0027] In one method according to the present invention, hair is
provided, preferably washed and unbleached. The hair can be
harvested from a human or animal source. The patient or a human
donor is a preferred source of hair for some medical applications,
as hair from these sources is most likely to result in a
non-antigenic product, although animal hair may be acceptable for
certain individuals that do not have animal product allergy
problems. In one method, the hair is washed with Versa-Clean.TM.
(Fisher Scientific, Pittsburgh, Pa.), rinsed with deionized water,
and allowed to air dry. Partial Oxidation of Keratin
[0028] The hair can be oxidized in peracetic acid or another
suitable reagent such as H.sub.2O.sub.2. A preferable treatment
utilizes from 1% to 32% peracetic acid, at a temperature between
about 0.degree. C. and 100.degree. C. for between 0.5 and 24 hours.
One method treats 30 grams of hair with 500 mL of 32% peracetic
acid at 4.degree. C. for 24 hours. This treatment with peracetic
acid partially oxidizes the naturally occurring disulfide linkages
to produce a protein with cysteic acid (--CH.sub.2SO.sub.3H)
residues and remaining disulfide linkages. The hair is recovered,
preferably by filtration through a coarse fritted glass filter, and
rinsed numerous times with deionized water until the rinse solution
has a pH of 6.0 or higher. The hair can then be dried in a vacuum
oven at between 20.degree. C. and 50.degree. C. for between 0.5 and
5 days. One method dries the hair in a vacuum oven at 40.degree. C.
for several days. The dried hair can then be pulverized and ground
into a fine powder. One method of grinding the hair uses a ceramic
mortar and pestle.
[0029] Reduction of Partially Oxidized Keratin
[0030] The keratin powder can be suspended in ammonium
thioglycolate. In one method, pulverized keratin powder, derived
from hair as described above, is suspended in about 3N ammonium
hydroxide containing ammonium thioglycolate. About 6 grams of
keratin powder can be added per 75 mL of ammonium hydroxide. The
strength of ammonium hydroxide is preferably about 3N, and the
preferred concentration of ammonium thioglycolate is about 11 mL
(as thioglycolic acid) per 75 mL of ammonium hydroxide. The
suspension can then be heated for a time sufficient to solubilize
the soluble fraction of the hair. The suspension, in one method, is
heated between 50.degree. C. and 90.degree. C. for between 1 and 24
hours, followed by cooling. According to another method, the
suspension is heated to about 60.degree. C. for about 4 hours and
cooled to room temperature. This treatment cleaves the remaining
disulfide linkages to produce cysteine residues in the protein
structure. At this point, the keratin protein contains both cysteic
acid residues and cysteine residues. The ratio of cysteic acid
residues and cysteine residues can be controlled by varying the
time, temperature, and concentration of oxidant in the peracetic
acid treatment step previously described. The presence of sulfonic
acid residues imparts a hydrophilic property to the hair, as well
as the final sheet product.
[0031] Separation of Partially Oxidized Reduced Keratin
[0032] After the oxidation/reduction treatment described above, a
resistant keratin fraction remains, consisting primarily of beta
keratin. This keratin fraction is preferably at least 80% beta
keratin, most preferably greater than about 90% beta keratin. This
fraction is insoluble in the suspension and is removed in one
method by centrifugation at about 10,000 g for about 10 minutes. A
thick, jelly-like supernatant remains and is discarded or, more
preferably, kept for another use. The remaining insoluble fraction
is composed mostly of the original cuticle (outer layer of hair
shaft) and is composed primarily of beta keratin.
[0033] Acidification of Partially Oxidized Reduced Keratin
[0034] The insoluble material is transferred to another container
and acidified to a low pH. The pH is preferably less than about 3
and most preferably less than about 1. In one method the pH is less
than about 1 and the acid used can be either concentrated sulfuric
acid or formic acid. This treatment disrupts hydrogen bonding of
the cuticle structure of the hair shaft. The low pH disrupts the
hydrogen bonds responsible for tightly binding the keratin protein,
resulting in its resistance to chemical modification. Applicants
believe the acid at least partially unfolds or swells the protein,
enhancing the solubility. The slurry preferably has a concentration
in the range of 0.001 grams/mL to 0.6 grams/mL. The slurry most
preferably has a concentration in the range of 0.2 grams/mL to 0.3
grams/mL.
[0035] Neutralization, Concentration and Oxidation of Partially
Oxidized, Reduced Keratin
[0036] The unfolded or swelled keratin slurry can then be made
slightly basic with ammonium hydroxide, preferably about 6N
strength. The slurry can then be cast onto a flat surface and air
dried to produce the cross-linked sheet. A preferred relative
humidity range for drying is between 0% and 90%. The relative
humidity is most preferably between about 40% and 60% relative
humidity. The partially unfolded, swelled, and partially
solubilized keratin refolds upon addition of the base during
drying, causing hydrogen bonding of the keratin. The free thiol
groups form disulfide linkages.
[0037] The insoluble keratin fraction from hair is thus treated so
as to have both sulfonic acid groups and thiol groups, and is
separated from the soluble fraction. The insoluble fraction is
treated with acid to partially unfold, swell, and solubilize the
keratin, followed by treatment with base and casting onto a flat
surface to refold the protein and form some disulfide bonds.
[0038] In an alternate method, in the acidification step, the
keratin is suspended in a volatile acid, such as formic acid,
having sufficiently low pH to partially unfold or swell the keratin
protein. In this method, the treatment with volatile base can be
dispensed with. The acidification step can be immediately followed
by forming the keratin slurry into a sheet.
[0039] The resulting sheet may be cleansed of soluble reagents by
repeated treatment with hot (boiling), deionized water, yielding a
cross-linked, pure keratin sheet. The moist keratin sheet, formed
of keratin derived primarily from beta keratin, has the consistency
of moist paper. The sheet produced will dry to a brittle material
which can be rehydrated to a supple skin-like material, suitable
for use as a sheet wound dressing. The sheet retains water and the
rehydrated sheet has the look and feel of skin. In a preferred
method of use, the sheet is hydrated sufficiently to allow the
sheet to be draped over a wound.
[0040] Keratin Slurry Including Partially Oxidized Alpha and Beta
Keratin Fractions
[0041] In an alternate embodiment of the present invention, the
keratin centrifugation step used to separate the soluble and
insoluble partially oxidized keratin fractions is omitted and both
fractions are used in further processing. In one embodiment, both
fractions are further processed together with acid as described
above. In this method, both soluble and insoluble fractions are
transferred to another container and acidified to a low pH. The
unfolded or swelled keratin slurry can then be made slightly basic
with ammonium hydroxide, preferably about 6N strength. The slurry
can then be cast onto a flat surface and air dried to produce a
cross-linked sheet. As an alternate method, in the acidification
step, the keratin is suspended in a volatile acid, such as formic
acid, as described previously. In this method, the treatment with
volatile base can be dispensed with. In one method, the thick
slurry having both keratin fractions can be cast into a thin film
as previously discussed. The resulting product has a somewhat
smoother texture than a pure beta keratin derived product. In
another method, the thick slurry can be further concentrated and
used to form a bulk keratin product as discussed below.
[0042] Use of Partially Oxidized Alpha Keratin Fraction
[0043] In one embodiment, the keratin in the keratin solution is at
least 90% keratin derived from alpha keratin. The resulting keratin
solution containing partially oxidized keratin derived primarily
from alpha keratin can be utilized as described above, in the
formation of films and sheets. The alpha keratin found in hair is
primarily crystalline prior to processing, but is primarily
amorphous after processing and cross-linking. Thus the terms
"alpha" and "beta" refer to the keratin protein structures at the
source, not necessarily the keratin protein structures after
processing and cross-linking. The alpha keratin is derived
primarily from hair cortex keratin while the beta keratin is
derived primarily from hair cuticle keratin. A preferred method
utilizes hair cortex keratin.
[0044] In another embodiment, the soluble keratin fraction is used
to form a sheet or film. After centrifugation such as described
above, the insoluble fraction can be set aside for other use. A
thick, jelly-like supernatant remains, which includes a soluble,
partially oxidized keratin fraction derived primarily from alpha
keratin. The keratin fraction is termed "soluble" as it is soluble
in a basic, aqueous solution. In a preferred method, "soluble"
keratin refers to a keratin fraction soluble at a pH of 10 or
greater, but which may be soluble at lower, basic pH. In a
preferred method, "insoluble" keratin refers to keratin insoluble
at a pH of 10. The supernatant is collected. The supernatant can be
treated with concentrated HCl until a gummy precipitate is
produced. The precipitate can be collected, washed with deionized
water, and dissolved in 15 mL of 3N ammonium hydroxide, forming a
keratin solution.
[0045] Keratin Sheet Applications
[0046] Applicants believe the keratin product made according to
this method is suitable for use as a cell-growth scaffold that is
mitogenic and as a nutrient support for cell growth. Applicants
also believe the cross-linked keratin sheet can be used as a
scaffold material for a variety of cells, including skin component
cells (keratinocytes, fibroblasts, endothelial cells), osteoblasts,
chondrocytes, and hepatocytes. In particular, applicants have shown
that skin component cells will grow and proliferate favorably on
the keratin sheet. Applicants further believe the keratin sheet can
be used as a diffusion membrane and to encapsulate cells for
various applications.
[0047] Anti-bacterial additives, ointments, and biologicals such as
growth factors or collagen can be added to the keratin sheet.
Bactericidal ointment or a suspension of antibiotics or biologicals
can be impregnated into the sheet dressing by passing a blade
having the additive at its front over the sheet, thereby evenly
distributing the additive over the sheet. Alternatively, the sheet
material can be soaked in a solution containing the desired
additive and the additive allowed to precipitate onto the surface
of the sheet. The solvent can then be flashed off, leaving the
sheet material impregnated and coated with the desired
additive.
[0048] Keratin Reduction Without Previous Partial Oxidation
Step
[0049] Clean, keratin-containing hair prepared as previously
described can be suspended in a reducing agent. A preferred
reducing agent is ammonium thioglycolate. Other reducing agents
believed suitable for use in the present invention include
mercaptoethanol, dithiothreitol, thioglycerol, thiolactic acid,
glutathione, cysteine, and sodium sulfide. In one method, the
washed and cut hair, as described above, is suspended in about 3N
ammonium hydroxide containing ammonium thioglycolate. The ammonium
hydroxide is believed to deprotonate the carboxylic acids and the
cysteine thiol groups, forming a polyanionic polymer having
increased solubility in water. The ammonium hydroxide is believed
to partially swell the keratin protein, exposing additional
disulfide linkages to reaction with thioglycolic acid. About 6
grams of hair can be added per 75 mL of ammonium hydroxide. The
strength of ammonium hydroxide is preferably about 3N and the
preferred concentration of ammonium thioglycolate is about 11 mL
(as thioglycolic acid) per 75 mL of ammonium hydroxide. The
suspension can then be heated for a time sufficient to solubilize
the soluble fraction of the hair. The suspension in one method is
heated between 50.degree. C. and 90.degree. C. for between 1 and 24
hours, followed by cooling. In a preferred method, the suspension
is heated to about 60.degree. C. for about 2 hours under a nitrogen
atmosphere and homogenized with a tissue homogenizer, as will be
described in detail in the next section, for about 30 minutes until
a fine dispersion is produced.
[0050] Homogenizing/Comminuting of Reduced Keratin
[0051] Homogenizing, as the term is used herein, refers to the hair
particles being comminuted, that is, broken down into smaller
particles using a rotor/stator combination homogenizer blade. The
reduced hair was homogenized in situ in the ammonium thioglycolate
solution using procedures described in U.S. Pat. No. 5,358,935
(without using liquid N2) incorporated by reference above. Hair is
protected by a tough outer keratin layer resistant to chemical
treatment. The outer layer is formed of primarily beta keratin
material. The homogenizing separates the outer, protective cuticle
material from the inner, cortex material and comminutes the hair to
make small keratin particles. The cortex contains keratins
moderately soluble in water, but keratins not normally exposed to
water, lying within the protective cuticle. The cortex contains
primarily alpha keratin. The homogenization also exposes disulfide
bonds to reactants such as thioglycolate. The dispersion in one
method is further heated an additional two hours at 60.degree. C.
under a nitrogen atmosphere before being cooled to room
temperature. The continued heating step provides time for the
ammonium thioglycolate to break and reduce the newly exposed
cysteine disulfide linkages. A thick slurry is the expected result
in a preferred method. The heating speeds up the reduction of
disulfide bonds. The nitrogen atmosphere prevents the oxidation of
thiol groups by atmospheric oxygen. Applicants believe this
treatment cleaves disulfide linkages to produce cysteine and
cysteinethioglycolate disulfide residues in the protein
structure.
[0052] Separation
[0053] After the treatment described above, a keratin fraction
resistant to the treatment remains, consisting primarily of beta
keratin. This fraction is insoluble in the suspension and is
removed in one method by centrifugation at about 10,000 g for about
10 minutes. The insoluble fraction can be set aside for other use.
A supernatant remains, which includes a soluble keratin fraction
derived primarily from alpha keratin. The keratin fraction is
termed "soluble" as it is soluble in a basic, aqueous solution. In
a preferred method, "soluble" keratin refers to a keratin fraction
soluble at a pH of 10 or greater, but which may be soluble at
lower, basic pH. In a preferred method, "insoluble" keratin refers
to keratin insoluble at a pH less than 10. The supernatant is
collected. The supernatant can be treated with concentrated HCl
until a gummy precipitate is produced. The precipitate can be
collected, washed with deionized water, and dissolved in 15 mL of
3N ammonium hydroxide, forming a keratin solution.
[0054] In one embodiment, the keratin in the keratin solution is at
least 90% keratin derived from alpha keratin. The alpha keratin
found in hair is primarily crystalline prior to processing but is
primarily amorphous after processing and cross-linking. Thus the
terms "alpha" and "beta" refer to the keratin protein structures at
the source, not necessarily the keratin protein structures after
processing and cross-linking. The alpha keratin is derived
primarily from hair cortex keratin while the beta keratin is
derived primarily from hair cuticle keratin. Hair cuticle keratin
typically includes substantial color from the original hair. Hair
cortex keratin does not include the original hair color. A
preferred method utilizes hair cortex keratin.
[0055] Film and Sheet Formation
[0056] The solution can be cast into a thin film and allowed to air
dry into a cross-linked film derived primarily from alpha keratin.
The keratin re-forms disulfide bonds, giving the film added
strength. Weaker bonds, such as hydrogen bonds, also impart
strength to the keratin-based film as the solution becomes more
concentrated, bringing the keratin proteins in closer proximity to
one another. In one method, the solution is poured onto a flat
surface, for example a glass surface. In another method, the
solution is poured onto a rotating drum or moving belt. Pouring the
solution onto a flat surface produces a thin, flat geometry
resembling that of the final film. Forming the flat surface also
creates a high surface to volume ratio, allowing air to penetrate
into the solution a substantial fraction of the total depth and
volume.
[0057] Concentration and Oxidation
[0058] Air drying performs several functions. First, the air
removes water, thereby concentrating the keratin solution. The more
concentrated solution increases the rate of formation and number of
re-formed disulfide bonds. The disulfide bonds formed or reformed
are not necessarily between the same cysteine groups in the initial
protein. Second, the air contains oxygen, which oxidizes the free
thiol groups in the protein, forming disulfide bonds. Other
oxidizing gases can be used in place of air, for example oxygen.
Oxidizing liquids such as hydrogen peroxide are also suitable for
oxidizing the free thiol groups. Third, the air allows the ammonium
hydroxide to evaporate. The resulting lowered pH also helps reform
the disulfide bonds. Fourth, the air allows some excess
thioglycolate to escape. When the film formation is carried out in
the presence of nitrogen rather than air, applicants believe the
film formed has far fewer disulfide bonds, but that the film is
bound with hydrogen bonds, resulting in a film that is softer than
the film formed in the presence of oxygen. The resulting residual
thiol activity would provide sites for the incorporation of
desirable thiol-containing biological factors.
[0059] The concentration and oxidation causes the formation of a
tough, insoluble material. Excess thioglycolate, and the disulfide
of thioglycolic acid, may remain in the film and can be removed
through extraction in boiling water. In one method of cleaning, the
film is immersed in boiling water for about 1.5 hours, changing the
water every 15 minutes. This cleaning is believed to remove mostly
excess, unreacted thioglycolate as opposed to thioglycolate bound
to the protein backbone.
[0060] Slow evaporation can also be used to remove ammonium
hydroxide from the material, thereby lowering the pH and promoting
cross-link formation. Reducing pH in itself causes increased
cross-linking and precipitation of protein. An additional
crosslinking agent such as glutaraldehyde can be used to form
cross-links other than disulfide cross-links. The use of
glutaraldehyde allows cross-linking without requiring the same
degree of concentration or water removal as required for
cross-linking relying primarily on disulfide bond formation and
could also increase the final degree of crosslinking over the
oxidation crosslinking procedure.
[0061] Another method for forming the disulfide cross-links
includes the steps of removing the water and ammonium hydroxide
under vacuum. In one method, the soluble keratin fraction in
solution is placed into a chamber and a vacuum pulled on the
chamber, removing much of the water from the material. The water is
volatilized at a low temperature, leaving behind a cross-linked
keratin material
[0062] Uses
[0063] Applicants believe the resulting material can be formed into
a thin film, wound dressing, or tissue-engineering scaffold.
Another use is as a diffusion membrane, for example, for drug
delivery. Yet another use is for coating implantable devices, such
as stents and maxillofacial implants, with the non-antigenic
cross-linked keratin film material. The parts to be coated can be
dipped in the keratinous solution, followed by air drying or other
method to promote cross-linking. This gives strong adherence to the
implant since cross-linking occurs on the actual implant shape as a
thin film. Yet another use is as an encapsulant to encapsulate
cells. Individual cells can be encapsulated, allowing, for example,
the film to act as a nutrient supply, a mitogen, or a diffusion
membrane.
[0064] Further Concentration
[0065] In another method embodying the present invention, the
resulting keratin suspension is further concentrated. The resulting
solution is preferably concentrated to a concentration of between
about 0.1 and 0.5 grams per mL, more preferably between about 0.3
and 0.4 grams per mL, and most preferably about 0.35 grams per mL.
The concentrated keratin solution can be used to create a porous,
open cell keratin scaffold, discussed in detail below, in the open
cell section.
[0066] Keratin Slurry Including Alpha and Beta Keratin
Fractions
[0067] In an alternate embodiment of the present invention, the
keratin centrifugation step used to separate the soluble and
insoluble keratin fractions is omitted, and both fractions are used
in further processing. In one method, the thick slurry having both
keratin fractions can be cast into a thin film as previously
discussed. The resulting product has a somewhat rougher texture
than the pure alpha keratin derived product. In another method, the
thick slurry can be further concentrated and used to form a bulk
keratin product as previously discussed.
[0068] Keratin Slurry Including Alpha and Beta Keratin Fractions
with Further Acid Treatment
[0069] In another embodiment of the present invention, the keratin
centrifugation step used to separate the soluble and insoluble
keratin fractions is omitted, and both fractions are further
processed with acid. In this method, both soluble and insoluble
fractions are transferred to another container and acidified to a
low pH. The pH is preferably less than about 3 and most preferably
less than about 1. In one method, the pH is less than about 1, and
the acid used can be hydrochloric, concentrated sulfuric, or formic
acid. Applicants believe the acid at least partially swells the
protein, enhancing the solubility of the insoluble fraction. The
slurry preferably has a concentration in the range of 0.001
grams/mL to 0.6 grams/mL. The slurry most preferably has a
concentration in the range of 0.2 grams/mL to 0.3 grams/mL.
[0070] The unfolded or swelled keratin slurry can then be made
slightly basic with ammonium hydroxide, preferably about 6N
strength. The slurry can then be cast onto a flat surface and air
dried to produce the cross-linked sheet. A preferred relative
humidity range for drying is between 0% and 90%. The relative
humidity is most preferably between about 40% and 60% relative
humidity. The partially unfolded or swelled, partially solubilized
keratin refolds upon addition of the base during drying, causing
hydrogen bonding of the keratin. The free thiol groups form
disulfide linkages. In an alternate method, glutaraldehyde can be
added to the partially solubilized keratin to provide an increased
degree of crosslinking. As an alternate method, in the
acidification step, the keratin is suspended in a volatile acid,
such as hydrochloric or formic acid, having sufficiently low pH to
partially swell the keratin protein. In this method, the treatment
with volatile base can be dispensed with. The acidification step
can be immediately followed by forming the keratin slurry into a
sheet. The keratin slurry can also be further concentrated for
production of bulk keratin.
[0071] Keratin Slurry Including Primarily Beta Keratin with Further
Acid Treatment
[0072] In another embodiment of the present invention, the keratin
centrifugation step used to separate the soluble and insoluble
keratin fractions is performed and the beta fraction is further
processed with acid. In this method, the insoluble fraction is
transferred to another container and acidified to a low pH. The pH
is preferably less than about 3 and most preferably less than about
1. In one method, the pH is less than about 1 and the acid used can
be hydrochloric, concentrated sulfuric, or formic acid. Applicants
believe the acid at least partially swells the protein, enhancing
the solubility of the insoluble fraction. The slurry preferably has
a concentration in the range of 0.001 grams/mL to 0.6 grams/mL. The
slurry most preferably has a concentration in the range of 0.2
grams/mL to 0.3 grams/mL.
[0073] The keratin slurry can then be made slightly basic with
ammonium hydroxide, preferably about 6N strength. The slurry can
then be cast onto a flat surface and air dried to produce the
cross-linked sheet. A preferred relative humidity range for drying
is between 0% and 90%. The relative humidity is most preferably
between about 40% and 60% relative humidity. The partially
unfolded, swelled, partially solubilized keratin refolds upon
addition of the base during drying, causing hydrogen bonding of the
keratin. The free thiol groups form disulfide linkages. In an
alternate embodiment, glutaraldehyde can be added to the partially
solubilized keratin to provide an increased degree of crosslinking.
As an alternate method, in the acidification step, the keratin is
suspended in a volatile acid, such as formic acid, having
sufficiently low pH to partially swell the keratin protein. In this
method, the treatment with volatile base can be dispensed with. The
acidification step can be immediately followed by forming the
keratin slurry into a sheet. The keratin slurry can also be further
concentrated for production of bulk keratin.
[0074] Keratin Open Cell and Bulk Materials
[0075] The present invention also includes methods for forming
keratin bulk materials and porous open cell materials. The bulk
material is suitable for use as a cross-linked implantable device,
which can be used for maxillofacial restoration, for example, for
soft and hard tissue replacement. The bulk material can also be
used for orthopedic applications such as bone filler and cartilage
regeneration. A tubular form of the implanted material can also be
used for neurological applications such as nerve regeneration
guides. The porous keratin material can be used as a
tissue-engineering scaffold.
[0076] The invention includes processes for forming solid and
porous bulk keratin materials. A keratinous material, such as human
hair, is provided. The hair is suspended in liquid and reduced with
a reducing agent, breaking the disulfide bonds. A keratinous slurry
is the preferred result. The slurry, which can be further processed
and purified, is preferably further concentrated and deposited into
a mold to form a solid part in the shape of the mold.
Alternatively, the slurry can be used to process open cell, foam
materials using a variety of methods described in the literature.
One technique uses a spray of the atomized keratin solution on the
surface of a cooled mold, thereby building up a foam structure as
described by Lo et al. for PLLA foam fabrication (H. Lo, S.
Kadiyala, S. E. Guggino, and K. W. Leong, "Poly (L-lactic acid)
foams with cell seeding and controlled-release capacity," J.
Biomed. Mater. Res., Vol. 30, pp. 475-484, 1996). A second
technique, also developed for PLA/PGA polymers, uses freeze drying
emulsions of polymer solutions to process open cell polymer
structures (K. E. Healy, K. Whang, and C. H. Thomas, "Method of
fabricating emulsion freeze-dried scaffold bodies and resulting
products," U.S. Pat. No. 5,723,508, issued Mar. 3, 1998). A similar
process can be modified using the appropriate solvents and
conditions to make an open cell keratin scaffold. For example, the
keratin is dissolved in a volatile non-polar solvent and mixed with
a volatile polar solvent in which the keratin is insoluble. These
two solvents are immiscible. An emulsion is generated using
ultrasound or a homogenizer, frozen, and freeze-dried to remove the
solvents. An oxidizing agent, such as air or a peroxide or a
crosslinking agent such as glutaraldehyde, can be supplied to the
keratin material in the emulsion stage. The keratin concentration
and oxidizing agent act to promote keratin cross-linking. The
resulting keratin cross-linked product is hard and porous, with a
microstructure dependent on the exact method used.
[0077] Numerous characteristics and advantages of the invention
covered by this document have been set forth in the foregoing
description. It will be understood, however, that this disclosure
is, in many respects, only illustrative. Changes may be made in
details, particularly in matters of shape, size, choice of
reagents, and ordering of steps without exceeding the scope of the
invention. The invention's scope is, of course, defined in the
language in which the appended claims are expressed.
[0078] Experimental Results, Partially Oxidized Keratin Product
[0079] In a first experiment, a sheet wound dressing not requiring
a binder was prepared from keratin derived from human hair. Human
hair was obtained from males aged 10 to 30 years, washed with
Versa-Clean.TM. (Fisher Scientific, Pittsburgh, Pa.), rinsed with
deionized water and allowed to air dry. This hair was subsequently
chopped into approximately 0.25-inch to 2-inch lengths using
shears. Thirty grams of this hair was treated with 500 mL of 32%
peracetic acid (Aldrich Chemical, Milwaukee, Wis.) at 4.degree. C.
for 24 hours. This treatment partially oxidized the disulfide
linkages. The hair was recovered by filtration through a coarse
fritted glass filter and rinsed numerous times with deionized water
until the rinse solution was pH 6.0 or higher. The hair was dried
under vacuum at 40.degree. C. for several days until completely dry
and ground to a fine powder with a ceramic mortar and pestle. The
resulting material, 19 grams, was further modified to produce a
flexible, hydratable sheet composed primarily of beta keratin.
[0080] Six grams of the pulverized, oxidized hair was suspended in
75 mL of 3N ammonium hydroxide containing 11 mL of ammonium
thioglycolate (as thioglycolic acid). The suspension was heated to
60.degree. C. for 4 hours and then cooled to room temperature. This
treatment cleaved the remaining disulfide linkages to produce
cysteine residues in the protein structure. An insoluble fraction
remained, which was resistant to solubilization by the ammonium
hydroxide and ammonium thioglycolate. The insoluble fraction,
comprised mostly of beta keratin, was isolated by centrifugation at
10,000 g for 10 minutes. A thick, jelly-like supernatant was
removed from the centrifuged material and set aside.
[0081] The remaining insoluble fraction is composed mostly of the
original cuticle (outer layer of hair shaft) and is composed
primarily of beta keratin. The insoluble material was transferred
to a flask and acidified to a pH of between 0 and about 1 with
concentrated sulfuric acid. The partially unfolded keratin was made
slightly basic with 6N ammonium hydroxide. The slurry was then cast
onto a flat surface and air dried to produce a cross-linked sheet.
The resulting sheet was purified by immersion in boiling water,
which removed soluble reagents.
[0082] The use of keratin-containing materials in promoting wound
healing was demonstrated in several experiments. In a first
experiment, processed human hair was incubated with cell culture
media. The media/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
cultures using this media extract. Significant proliferation of
these wound healing cells was measured. Keratinocytes proliferated
profusely, fibroblasts proliferated modestly, and endothelial cells
proliferated profusely.
[0083] The mitogenic activity observed in fibroblast, keratinocyte,
and endothelial cell cultures is additional evidence that the
keratinous protein material is not only biocompatible, but also
mitogenic with these cell lines. Additional biocompatibility was
observed when keratin microfibrils were observed microscopically to
be in direct contact with cells in the cell cultures. Specifically,
keratinocytes and fibroblasts were observed to adhere to and
congregate around microfibrils, indicating that desirous cell
activity can be sustained on this naturally derived biopolymer
matrix.
[0084] In a second experiment, processed human hair powder was
incubated with cell culture media. The media/keratin mixture was
passed through a micro filter. This media extract was used in
proliferation studies with lymphocytes. The lymphocyte cell line
did not proliferate, indicating the material to be
non-immunogenic.
[0085] In a third experiment, processed human hair powder was
incubated with cell culture media. The media/hair mixture was then
passed through a micro filter. This media extract was used in
proliferation studies with activated T-lymphocytes. The
T-lymphocytes proliferated normally, indicating no inhibition of
the normal cell mediated immune response by the keratin. This
demonstrated no inhibition of this very important function of
immune cells.
[0086] In a fourth experiment, twenty-eight hairless rats were
wounded on either side of the dorsal midline with a dermatome,
creating a partial thickness wound 0.12 inches in depth, and
2.0.times.4.0 cm in surface area. Half the wounds were treated with
keratin powder, half were not, and both halves were covered with
polyurethane dressing. The wounds were observed for healing and
biopsied at days 0, 2, 4 and 6 for histochemical analysis.
Planimetry studies showed 97% epithelialization of the
keratin-treated wounds and 78% epithelialization of the non-treated
wounds at day 4. Histological analysis by H & E stain revealed
total epithelialization microscopically of the keratin-treated
wounds at day 2 and only partial epithelialization of the
non-treated wounds at day 2. Histological analyses at days 4 and 6
also revealed an acceleration of the epithelialization maturation
process in the keratin-treated wounds.
[0087] Human clinical studies are currently being performed on
donor sites for skin grafts. One half of the donor wound site is
treated with sterilized keratin powder and the opposite half
treated in a standard fashion, with Adaptic.TM. non-adhering
dressing from Johnson & Johnson. Preliminary results show the
keratin-treated halves epithelialize sooner and mature more
rapidly. This was confirmed through both clinical observations and
histological results of four-millimeter punch biopsies.
Subjectively, patients also have much less pain in the
keratin-treated wounds.
[0088] Experimental Results, Keratin Product Without Partial
Oxidation
[0089] In a fifth experiment, human hair was obtained from males
aged 10 to 30 years, washed with Versa-Clean.TM. (Fischer
Scientific, Pittsburgh, Pa.), rinsed with deionized water and
allowed to air dry. This hair was subsequently chopped into
approximately 0.25" to 2" lengths using shears. Six grams of hair
was suspended in 75 mL of 3N ammonium hydroxide containing 11 mL of
ammonium thioglycolate. This treatment cleaved the disulfide
cystine linkages to produce cysteine residues in the protein
structure. The suspension was heated to 60.degree. C. for 2 hours
under a nitrogen atmosphere and then homogenized with a tissue
homogenizer for 30 minutes until a fine dispersion was produced.
The dispersion was heated an additional 2 hours at 60.degree. C.
under a nitrogen atmosphere and then cooled to room temperature.
The thick slurry was transferred to a tube and centrifuged at 5000G
for 10 minutes. The supernatant was treated with concentrated
hydrochloric acid until a gummy precipitate was produced. The
precipitate was collected, washed with deionized water and then
dissolved in 15 mL of 3N ammonium hydroxide.
[0090] This solution was then cast into a thin film and allowed to
air dry. The solution was also further concentrated through
evaporation, and cast into a solid block of material. The removal
of the volatile base and water from the solution and the action of
the air upon the free thiol fraction of the soluble polypeptide
caused the material to crosslink into an insoluble, tough material.
The material was then purified and freed of any remaining
thioglycolic acid by extraction in boiling water for 1.5 hours.
[0091] In a sixth experiment, human hair was chemically treated as
previously described. This produced a keratin solution that was
then cast into a sheet and oxidatively cross-linked to produce a
non-soluble sheet of keratin. The sheet was purified by extraction
with boiling water for 1.5 hours, changing the water every 15
minutes. Segments of the sheeting were then incubated with
keratinocytes, fibroblasts, and human microvascular endothelial
cells. These cells were shown to grow and proliferate favorably on
the keratin sheet. This indicates that skin component cells
proliferate favorably in the presence of keratin sheeting produced
by the above-described method.
[0092] Numerous characteristics and advantages of the invention
covered by this document have been set forth in the foregoing
description. It will be understood, however, that this disclosure
is, in many respects, only illustrative. Changes may be made in
details, particularly in matters of shape, size and ordering of
steps without exceeding the scope of the invention. The invention's
scope is, of course, defined in the language in which the appended
claims are expressed.
* * * * *