U.S. patent application number 09/736957 was filed with the patent office on 2001-07-26 for keratin-based hydrogel for biomedical applications and method of production.
Invention is credited to Blanchard, Cheryl R., Smith, Robert A., Timmons, Scott F..
Application Number | 20010009675 09/736957 |
Document ID | / |
Family ID | 25526898 |
Filed Date | 2001-07-26 |
United States Patent
Application |
20010009675 |
Kind Code |
A1 |
Blanchard, Cheryl R. ; et
al. |
July 26, 2001 |
Keratin-based hydrogel for biomedical applications and method of
production
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.
Inventors: |
Blanchard, Cheryl R.;
(Warsaw, IN) ; Timmons, Scott F.; (San Antonio,
TX) ; Smith, Robert A.; (Jackson, MS) |
Correspondence
Address: |
Timothy S. Corder
VINSON & ELKINS L.L.P.
2300 FIRST CITY TOWER
1001 FANNIN STREET
HOUSTON
TX
77002-6760
US
|
Family ID: |
25526898 |
Appl. No.: |
09/736957 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09736957 |
Dec 12, 2000 |
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09365699 |
Aug 2, 1999 |
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6159496 |
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09365699 |
Aug 2, 1999 |
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08979456 |
Nov 26, 1997 |
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5932552 |
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Current U.S.
Class: |
424/445 ;
514/1.1; 514/21.2 |
Current CPC
Class: |
C07K 14/4741 20130101;
C08H 1/06 20130101; A61L 27/52 20130101; Y10S 530/842 20130101;
A61Q 19/00 20130101; A61L 31/047 20130101; A61L 27/34 20130101;
A61L 27/34 20130101; A61L 31/047 20130101; A61K 38/39 20130101;
A61L 27/227 20130101; Y10S 514/944 20130101; A61L 31/10 20130101;
A61L 26/008 20130101; A61L 26/0047 20130101; A61K 8/65 20130101;
A61L 27/34 20130101; A61K 38/1709 20130101; A61K 8/042 20130101;
A61L 27/227 20130101; A61L 27/60 20130101; A61L 31/10 20130101;
C08L 89/00 20130101; C08L 89/04 20130101; C08L 89/04 20130101; C08L
89/04 20130101; C08L 89/00 20130101 |
Class at
Publication: |
424/445 ;
514/21 |
International
Class: |
A61L 015/00; A61K
038/00 |
Claims
What is claimed is:
1. A tissue engineering cell scaffold for engineering bone tissue
comprising a keratin hydrogel comprising reformed
keratin-to-keratin disulfide links.
2. A tissue engineering cell scaffold in accordance with claim 1,
wherein the keratin hydrogel is derived primarily from human
hair.
3. A tissue engineering cell scaffold in accordance with claim 1,
wherein said keratin includes keratin protein bound together with
covalent, disulfide links, and said keratin protein has ionic
groups responsible for the hydrophilic property.
4. A tissue engineering cell scaffold in accordance with claim 1,
wherein said hydrogel has a concentration in the range of 0.05 to
0.4 grams of keratin per mL.
5. A tissue engineering cell scaffold for engineering cartilage
tissue comprising a keratin hydrogel comprising reformed
keratin-to-keratin disulfide links.
6. A tissue engineering cell scaffold in accordance with claim 5,
wherein the keratin hydrogel is derived primarily from human
hair.
7. A tissue engineering cell scaffold in accordance with claim 5,
wherein said keratin includes keratin protein bound together with
covalent, disulfide links, and said keratin protein has ionic
groups responsible for the hydrophilic property.
8. A tissue engineering cell scaffold in accordance with claim 5,
wherein said hydrogel has a concentration in the range of 0.05 to
0.4 grams of keratin per mL.
9. A method of engineering tissue comprising the implantation of a
tissue engineering scaffold comprising a keratin hydrogel
comprising reformed keratin-to-keratin disulfide links.
10. The method of claim 10, wherein said keratin hydrogel is
derived primarily from human hair.
11. The method of claim 10, wherein said keratin includes keratin
bound together with covalent, disulfide links, and said keratin has
ionic groups responsible for the hydrophilic property.
12. The method of claim 10, wherein said hydrogel has a
concentration in the range of 0.05 to 0.4 grams of keratin per mL.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/365,699, filed Aug. 2, 1999, issued as U.S.
Pat. No. 6,159,496, which is a continuation of U.S. patent
application Ser. No. 08/979,456, filed Nov. 26, 1997, issued as
U.S. Pat. No. 5,932,552. The present application is related to U.S.
Pat. No. 5,979,526, entitled KERATIN-BASED SHEET MATERIAL FOR
BIOMEDICAL APPLICATIONS AND METHOD OF PRODUCTION. The present
application is also related to U.S. Pat. No. 5,358,935, entitled
NONANTIGENIC KERATINOUS PROTEIN MATERIAL, both herein incorporated
by reference.
FIELD OF THE INVENTION
[0002] The present invention is related to wound dressing materials
and tissue engineering scaffolds. More specifically, the present
invention is related to a cross-linked keratin hydrogel.
BACKGROUND OF THE INVENTION
[0003] Chronic wounds can be caused by a variety of events,
including surgery, prolonged bedrest 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 reepithelization; 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 reepithelization. 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 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 and is non-toxic and non-antigenic, and
most important, delivers effective healing agents to the wound
site.
[0008] 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.
SUMMARY OF THE INVENTION
[0009] 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.
[0010] 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.
[0011] 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.degree. 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.
[0012] 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.
[0013] 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.
[0014] 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.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] In a method according to the present invention, hair is
provided, preferably washed and unbleached. The hair is harvested
from a human or animal source. The patient or a human donor is a
preferred source of hair, as hair from these sources is most likely
to result in a non-antigenic wound healing 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.
[0016] 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 is believed to partially oxidize the naturally occurring
disulfide linkages to produce a protein with cysteic acid
(--CH.sub.2SO.sub.3H) residues, and remaining disulfide
linkages.
[0017] The hair is recovered, preferably with 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.
[0018] The keratin powder can be suspended in ammonium
thioglycollate. In one method, pulverized keratin powder, derived
from hair as described above, is suspended in about 3N ammonium
hydroxide containing ammonium thioglycollate. About six 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 thioglycollate is about 11 mL
(as thioglycollic 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. 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 4 hours and cooled to
room temperature. Applicants believe this treatment cleaves the
remaining disulfide linkages to produce cysteine residues in the
protein structure. At this point, the keratin protein is believed
to contain cysteic acid, cysteine and cysteine-thioglycollate
disulfide 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 to the final
hydrogel product.
[0019] 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 is set aside for other use. A
thick, jelly-like supernatant remains which includes a soluble
keratin fraction. The supernatant is collected.
[0020] The supernatant is preferably purified, using a method such
as dialysis. A preferred method uses dialysis against running water
using a dialysis membrane (Spectra/Por TM) having a cutoff of about
8000 MW. The resulting solution is preferably concentrated to a
concentration of about 0.1 grams per mL.
[0021] The keratin in solution is now ready for cross-linking 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 peracids,
peroxy carbonates, ammonium sulfate peroxide, benzoyl peroxide, and
perborates. Hydrogen peroxide is preferably added to the keratin
solution at about 0.5% to about 1.0% w/v, mixed well, and allowed
to stand at room temperature for several days. A preferred standing
time is about 3 days. The freely flowing solution slowly thickens
and converts to a cross-linked hydrogel after about 72 hours.
[0022] The soluble keratin fraction from hair is thus partially
oxidized so as to have the protein backbones interconnected with
disulfide linkages and having sulfonic acid residues. The partially
oxidized keratin is treated with a reducing agent to cleave some or
all of the remaining disulfide bonds, forming thiol groups and
cysteine-thioglycollate disulfide groups and solubilizing more of
the keratin proteins. After removing the insoluble fraction, the
keratin is oxidized to allow the formation of disulfide
cross-links. Disulfide cross-links are thus reformed. As used
herein, the term "reformed" refers to cross-links broken and formed
later in time, where individual linkages later formed could be, but
are not necessarily, between the same amino acid cysteine
pairs.
[0023] A cross-linked, pure keratin hydrogel results. The hydrogel
has sulfonic acid groups which are hydrophilic and bind water
within the hydrogel. The number of sulfonic acid groups corresponds
to the degree of keratin oxidation in the partial oxidation
step.
[0024] Applicants believe the keratin product made according to
this method is suitable for use as a wound healing agent and as a
mitogenic cell growth scaffold for tissue engineering applications
and as a nutrient support for cell growth. It is also suitable for
skin care applications. Anti-bacterial additives, ointments and
biologicals such as growth factors or collagen can be added to the
keratin hydrogel.
EXPERIMENTAL RESULTS
[0025] A keratin-based hydrogel wound healing agent not requiring
binder material was prepared from keratin derived from human hair.
Human hair was obtained from males 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
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 used to produce a keratin
hydrogel.
[0026] Six grams of the pulverized, oxidized hair was suspended in
75 mL of 3N ammonium hydroxide containing 11 mL of ammonium
thioglycollate (as thioglycollic acid). The suspension was heated
to 60.degree. C. for 4 hours and then cooled to room temperature.
This treatment cleaved any remaining disulfide linkages to produce
cysteine and cysteine-thioglycollate disulfide residues in the
protein structure. An insoluble fraction remained which was
resistant to solubilization by the ammonium hydroxide and ammonium
thioglycollate. The insoluble fraction, believed to be 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, with the insoluble material set aside for
use in a related product.
[0027] The supernatant was dialyzed for 72 hours against running
water using a dialysis membrane with an 8000 MW cutoff (Spectra/Por
TM). The resulting solution was concentrated to 50 mL, in-vacuo at
sub-ambient temperature. The solution was treated with 3% hydrogen
peroxide added at a rate of 0.5% to 1.0% w/v mixed well and allowed
to stand at room temperature for 3 days. The freely flowing
solution slowly thickened and converted to a crosslinked hydrogel
after 72 hours. The hydrogel can be used as a wound healing agent
or a cell scaffold.
[0028] 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
culture using this media extract. Significant proliferation of
these wound healing cells was measured. Keratinocytes proliferated
profusely, fibroblasts proliferated modestly, and endothelial cells
proliferated profusely.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] In a fourth experiment, human hair was chemically treated.
This produced a keratin slurry that was then cast into a sheet and
chemically crosslinked to produce a nonsoluble sheet of keratin.
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.
[0033] In a fifth 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.
[0034] 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 nonadhering 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 4 millimeter punch biopsies. Subjectively, patients also
have much less pain in the keratin treated wounds.
[0035] 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.
* * * * *