CA1110971A

CA1110971A - Collagen skin dressing

Info

Publication number: CA1110971A
Application number: CA304,330A
Authority: CA
Inventors: Teruo Miyata
Original assignee: PIKOK INTERNATIONAL TRADING CO Ltd
Current assignee: PIKOK INTERNATIONAL TRADING CO Ltd
Priority date: 1977-06-09
Filing date: 1978-05-29
Publication date: 1981-10-20
Also published as: AR215499A1; MY8400233A; JPS6330023B2; HK43583A; BE867988A; CH641963A5; FR2393581B1; NO150585B; IT7824302A0; IT1098321B; GB1602340A; BR7803698A; JPS545019A; AT361129B; AU519348B2; HK43483A; DE2823620A1; NL7806067A; NO782005L; NO150585C

Abstract

ABSTRACT OF THE DISCLOSURE

Skin or wound dressings are prepared in gel or film form from collagen and/or chemically modified collagen.

Description

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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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g~l 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 - a _ ' :

g71 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:

-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 97~

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.

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.

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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. -, 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 .~

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.

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.

Sterile, freeze-dried collagen sheet was prepared by the method described in Example 4, except that the collagen concen-~ 71 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.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the preparation of porous collagen skin dressing which comprises forming a collagen gel, ex-truding the collagen gel into a coagulation bath, forming a sheet of collagen from the extruded gel, cross-linking the extruded collagen, and subjecting the cross-linked sheet to freeze-drying.

2. The process of claim 1 in which the cross-linking is carried out by tanning with glutaraldehyde.

3. The process of claim 1, which comprises the steps of:
a) treating a source of collagen with a proteolytic enzyme to form a telopeptide-poor, monomolecularly disper-sed atelocollagen extract, b) precipitating atelocollagen from the extract, c) purifying the precipitated atelocollagen by redis-solving and reprecipitation, d) converting the extracted, purified atelocollagen to a gel, e) extruding the atelocollagen gel through a circular extrusion nozzle in a coagulation bath, f) recovering atelocollagen in tubular form from the coagulation bath, g) slitting the tubular collagen longitudinally to form a collagen sheet therefrom, h) cross-linking the longitudinal sheet, and i) subjecting the cross-linked sheet to freeze-drying.

4. The process of claim 3 in which the cross-linking is carried out by tanning with glutaraldehyde.

5. The process of claim 1 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

6. The process of claim 2 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

7. The process of claim 3 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

8. The process of claim 4 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

9. A process for the preparation of collagen skin dressing of limited porosity which comprises forming a collagen gel, extruding the collagen gel into a coagulation bath, forming a sheet of collagen from the extruded gel, cross-linking the extruded collagen, partially air-drying the cross-linked sheet, and subjecting the partially air-dried sheet to freeze-drying.

10. The process of claim 9 in which the cross-linking is carried out by tanning with glutaraldehyde.

11. The process of claim 9 for the preparation of a collagen skin dressing in sheet form whose upper and lower surfaces possess differing porosity characteristics which comprises the steps of:
a) treating a source of collagen with a proteolytic enzyme to form a telopeptide-poor, monomolecularly disper-sed atelocollagen extract, b) precipitating atelocollagen from the extract, c) purifying the precipitated atelocollagen by redis-solving and reprecipitation, d) converting the extracted, purified atelocollagen to a gel, e) extruding the atelocollagen gel through a circular extrusion nozzle in a coagulation bath, f) recovering atelocollagen in tubular form from the coagulation bath, g) slitting the tubular collagen longitudinally to form a collagen sheet therefrom, h) cross-linking the longitudinal sheet, i) partially air-drying the cross-linked sheet, and j) freeze-drying the partially air-dried sheet where-by the upper surface of said sheet becomes more concentra-ted in ateloeollagen content than the lower surface thereof.

12. The process of claim 11 in which the cross-link-ing is carried out by tanning with glutaraldehyde.

13. The process of claim 9 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

14. The process of claim 10 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

15. The process of claim 11 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

16. The process of claim 12 wherein there is included at least one material selected from antibiotics and bacte-ricidal agents.

17. A skin dressing of petroleum jelly consistency comprising enzyme-solubilized acylated atelocollagen gel and at least one substance selected from antibiotics and bacte-ricides.

18. A skin dressing according to claim 17 in which the acylated atelocollagen is succinylated atelocollagen.

19. A skin dressing of petroleum jelly consistency comprising solubilized succinylated collagen gel and at least one material selected from bactericidal agents and antibiotics.

20. A method for the preparation of a collagen skin dressing in gel form which comprises the steps of:
a) treating a source of collagen with a proteolytic enzyme to form a telopeptide-poor, monomolecularly disper-sed atelocollagen extract, b) precipitating atelocollagen from the extract, c) reacting an aqueous dispersion of the atelocollagen extract with an acylating agent, d) separating acylated atelocollagen from the reaction mixture, and e) converting the acylated atelocollagen to a gel of petroleum jelly consistency.

21. The method according to claim 20 in which the acylating agent is succinic anhydride.

22. The method according to claim 20 in which the acylated atelocollagen is purified by solution and re-pre-cipitation prior to its conversion to a gel.

23. The method according to claim 21 in which the acylated atelocollagen is purified by solution and re-pre-cipitation prior to its conversion to a gel.

24. The method according to claim 20 in which there is incorporated into the gel at least one substance selected from antibiotics and bactericides.

25. The method according to claim 21 in which there is incorporated into the gel at least one substance selected from antibiotics and bactericides.

26. The method according to claim 22 in which there is incorporated into the gel at least one substance selected from antibiotics and bactericides.

27. The method according to claim 23 in which there is incorporated into the gel at least one substance selected from antibiotics and bactericides.