Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to skin dressings consisting of
collagen or chemically modified collagen in gel form, in porous
sheet form or in semiporous film type form. The invention also
relates to the production of such skin dressings by preparation
of sterile succinylated collagen gel, and by preparation of
collagen porous sheet made by successive processes of
extrusion of collagen gel into coagulation bath, tanning with
glutaraldehyde, and/or partial air-drying followed by freeze-
drying. Bactericidal agents or antibiotics may be impregnated
into the gel or sheet-type dressing.
A number of investigators including the present inventors
~; have suggested the use of collagen material as a skin, burn or
wound dressing. The feature of this invention, however,
; consists in the form of the collagen dressing and in the method
of producing such desired types.
The National Fire Protection Association reported in
1962 that approximately l,800~000 persons sustain burns yearly
and occupy over ll,000 hospital beds per day. There is a great
need for a readily available, easily stored and temporary
substitute for human skin for the effective treatment of
thermal burns and other forms of skin loss. It is common
practice to cover skin loss area with split-thickness autografts,
homografts and heterografts. Such treatments protect against
` infection, the loss of protein, fluid and electrolytes from
exposed tissue. These treatments, however have the following
drawbacks. Grafts are difficult to obtain, and to store for
any prolonged period of time and also are quite expensive.
These difficulties could be reduced by the development of
artificial skin dressings which are inexpensive and readily
available to use.
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Collagen is a major protein of connect tissue such as
skin, cornea, etc. and can be solubilized, separated and
purified by the treatment with proteolytic enzymes (other than
collagenase), e.g., proctase, pepsin, trypsin and pronase.
Solubilized collagen is telopeptides-poor, relatively inexpensive
and ideal as a material for development into a skin wound
dressing.
Solubilized collagen has many NH2 and COOH groups in its
structure, and chemical modifications of the molecule can be
readily made, e.g., all or some of the amino groups may be
acylated by reaction with a mixture of acetic anhydride and
acetic acid. Similarly, succinic anhydride reacts with
collagen replacing amino groups by carboxyl groups. The
carboxyl groups contained in the molecule are susceptible to
esterification by the standard reaction with acidified alochol,
e.g., reaction with anhydrous methanol acidified with HCl. In
the above reactions the net isoelectric point of collagen can
- be controlled, either negative or positive, or completely
neutralized.
All known types of collagen and chemically modified
collagen may be employed in the practice of this invention
e.g., native, denatured collagen (neutral isoelectric point);
esterified collagen (alkaline isoelectric point) and modified
amino-group forms e.g., anhydride derivatives (acidic lsoelectric
point). In the preparation of gel type dressings of this
invention the use of anhydride derivatives (acidic isoelectric
point) e.g., succinic anhydride derivatives of collagen are
preferred.
Gel Type
The gel type skin dressings of this invention are
especially suitable for application to irregular body surfaces
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e.g., areas of the joints, elbows, knees, etc. The gel dressing
is preferably used in the form of a viscous paste of petroleum
jelly consistency and contains 1 - 10%, preferably 2 - 5~ of
collagen.
In the preparation of the collagen gel, skin or hide is
solubilized in an enzyme solution at acidic pH. The resulting
gel is a viscous material which is recovered by filtering, e.g.,
through cheese cloth and/or a millipore filter. The viscous
solution is made alkaline by addition of caustic to a pH of
about 10. At this stage the material is permitted to stand in
order to inactivate any remaining enzyme. The material is
thereafter neutralized, the collagen collected by centrifuge
and washed with water. A second purification step follows,
namely, redissolving in aqueous acid (pH 2.0 - 5.0), reprecipi-
tation by neutralization to a pH of 6 to 7, and purification to
remove acid by dialysis against water. The neutral gel is
recovered and at this stage antibiotics or bactericides or
both may be added before storage of the gel material.
Collagen material used in the preparation of gel is
preferably not a multimer and, therefore, the material is not
- subjected to tanning during its preparation.
Succinylated collagen was preferably used for making
viscous gel skin dressing since it can be redissolved in water
at physiologic pH (6.8 - 7.4) without requiring dialysis against
water to remove acid during its preparation. Collagen concentra-
tion of gel skin dressing was between 0.5% to 7~ and into it
was incorporated the following bactericidal agents: Silver
nitrate solution (0.5 g/100 ml) or silver lactate solution
(9.5 g/100 ml) 25 mg/ml Lincomycin, 5 mg/ml Amphotericin B
and 25 mg/ml Gentamicin, or one or more thereof.
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Pourous Sheet Type
Another type of collagen skin dressing (porous sheet)
was prepared as follows: Solubilized collagen gel (pH 2.0 -
3.5, collagen concentration 1% - 10%) was extruded from a
tubular nozzle into coagulation bath (saturated NaCl).
Coagulated tubular collagen was cut longitudinally to obtain
sheet and tanned with 1 - 5~ glutaraldehyde in saturated NaCl
containing 0.05 M Na2HPO4 for 0.5 - 3.0 hours. Tanned collagen
sheet was washed with water repeatedly, then freeze-dried on
methylmethacrylate plate. To produce a semi-porous, film type
sheet in which the upper surface of the sheet is more concentrated
in collagen (resulting in an upper film type surface) and in
which the lower surface of the sheet is less concentrated in
collagen (i.e. more porous) the sheet is sub~ect to partial
air-drying prior to freeze-drying. Collagen sheet was sterilized
by ethylene oxide gas and soaked in a typical base solution
containing one or more bactericidal agents, such as silver
nitrate (0.5 g/100 ml), or silver lactate (0.5 g/100 ml), or
lactated Ringer's solution containing 25 mg/ml Gentamicin,
25 mg/ml Lincomycin, 25 mg/ml Colistimethate, 25 mg/ml Kanamycin,
and 5 mg/ml Amphotericin B; or lactated Ringer's solution
containing 25 mg/ml Lincomycin, 5 mg/ml Amphotericin B, and
25 mg/ml Gentamicin.
An effective skin dressing should have the following
properties:
1. good adherence to wound surface,
2. prevention of loss of protein, fluid and electrolytes,
3. prevention of infection,
4. reduction of pain,
5. no stimulation of local tissue response, etc.
Collagen skin dressings described here satisfy the above
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properties and are easy to use and less expensive. Gel skin
dressing is especially suitable for application to irregular
surfaces e.g., joint surfaces. Viscous gel-like paste and
gels of petroleum jelly consistency show excellent adherence
to wound surfaces. Porous sheet, and semi-porous film type
dressings also adhere firmly to the wound. Extensive cell
ingrowth into the porous collagen sheet was observed. All
skin dressings indicated effective protection against infection
and good wound healing.
The present invention may be further understood from the
following examples:
EXAMPLE 1
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Fresh calfskin (about 5 kg) was dehaired, cleaned by
shaving and cut into small pieces. The skin was washed
repeatedly with 10% NaCl containing a 0.2~ sodium azide
bactericide and with sterilized water. The skin was solubilized
in 10 liters of water (pH 2.5 HCl) containing 30 mg/ml
Gentamicin by addition of 1 g. of pepsin (approximate ratio of
enzyme to colla~en was 1/400) at 20C for 4 days with intermittent
stirring. The resulting viscous solubilized collagen was
filtered through cheesecloth, its pH raised to 10 by NaOH and
allowed to stand for 24 hours at 20C to inactivate the pepsin.
The pH of collagen was then adjusted to 7-8 (HCl) and collagen
precipitate was collected by centrifuging and washed with
sterilized water. The washed precipitate was redissolved in
acidic solution and reprecipitated at pH 7-8 for further
purification.
Succinylation of solubilized collagen was preformed as
follows: Ten grams (dry basis) of solubilized collagen
precipitate was resuspended in 4 liters of water and its pH
adjusted to 9.0 by NaOH. Acetone solution (100 ml) containing
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2.0 g of succinic anhydride was gradually added to the collagen
suspension. During the addition of succinic anhydride the pH
of collagen suspension was maintained at about 9.0 by NaOH
solution. Succinylated collagen was precipitated by acidification
to about pH 4.2, washed with water and freeze-dried. This
freeze-dried collagen (sponge-like in form) was sterilized to
remove micro-organisms. Five grams of the sterilized collagen
sponge was dissolved in acidified water. Upon addition of NaOH
to a pH of abo~t 7.4 the collagen emerges in a highly viscous
form. It was preserved by addition of silver nitrate (0.5%
solution). This gel skin dressing was excellent in adhesion
to the wound, in wound healing, and in protection against
infection.
EXAMPLE 2
Succinylated solubilized collagen was prepared by the
method described in Example 1. Sterilized succinylated collagen
(10 g) was treated in the same manner as in Example 1 but
preserved by Silver Lactate (0.5% solution). This gel skin
dressing again displayed excellent properties as above.
EXAMPLE 3
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Sterilized succinylated collagen was prepared by the
method described in Example 1 and treated in the same manner as
in Example 1 but was preserved in sterile lactated Ringer's
solution (0.6~ NaCl, 0.31% sodium lactate, 0.03% potassium
chloride, 0.02% calcium chloride, pH adjusted to 7.4) containing
the following antibiotics: 25 mg/ml Lincomycin, 5 mg/ml Ampho-
tericin B, and 25 mg/ml Gentamicin. This gel skin dressing once
again displayed excellent properties. -,
EXAMPLE 4
Solubilized collagen (not succinylated) was prepared by
the method described in Example 1. The collagen was dissolved
in dilute HCL solution (final pH 2.5, collagen concentration
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was 3~) and deairated under vacuum. Collagen acidic gel was
extruded into a coagulation bath (saturated NaCl) through an
appropriate nozzle. Coagulated tubing was recovered and cut
longitudinally to make it into sheets and tanned with 3%
glutaraldehyde in saturated NaCl containing 0.05 M Na2HPO4
for one hour. After repeated washing with water, collagen
sheet was freeze-dried on a plate of methylmethacrylate.
Freeze-dried sheets (10 cm x 10 cm) were sterilized by treatment
with ethylene oxide gas and preserved by soaking in 0.5~ silver
nitrate solution. Final thickness of the sheet was 3 mm. This
skin dressing was excellent in the adhesion to the wound, in
protection against fluid 105s and infection, and in wound
healing.
EXAMPLE 5
Collagen sheet was prepared by extrusion, tanning and
washing by the method described in Example 4, except using 5%
acidic collagen gel. Washed collagen sheet was then partially
air-dried on a plate of methylmethacrylate until the thickness
of the sheet became half of the original. This partial drying
reduces the porosity (collagen concentration higher) of the
upper surface of the sheet. It was then freeze-dried to render
the lower surface porous (collagen concentration lower), and
sterilized with ethylene oxide gas. Sterilized sheet was
preserved in sterilie 0.5~ silver lactate solution pH 7.4). The
final thickness of the sheet was 2 mm. This skin dressing had
finer porosity and greater strength than the sheet of Example 4.
It was excellent in protection of protein, fluid and electrolytes
loss, in protection against infection and in wound healing.
EXAMPLE 6
Sterile, freeze-dried collagen sheet was prepared by the
method described in Example 4, except that the collagen concen-
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tration was 5%. The sheet was preserved by soaking in sterile
lactated Ringer's solution (pH adjusted to 7.4) containing
the following antibiotics: 25 mg/ml Gentamicin, 25 mg/ml
Lincomycin, 25 mg/ml Colistimethate, 25 mg/ml Kanamycin and
S mg/ml Amphotericin B. The final thickness of the sheet was
4 mm. This sheet likewise displayed excellent skin dressing
properties.
