JP2005537882A - Wound dressing material containing a complex of anionic polysaccharide and silver - Google Patents

Abstract

The present invention provides a wound dressing material comprising a complex of anionic polysaccharide and silver. The material includes about 0.1 wt% to about 3 wt% silver. The present invention also includes wound dressings comprising such materials and the use of such materials in medicine.

Description

Detailed Description of the Invention

  The present invention relates to a wound dressing material comprising a complex formed of anionic polysaccharide and silver, such as oxidized regenerated cellulose (ORC), and its use for the treatment of wounds.

  Anionic polysaccharides such as alginate, hyaluronic acid and its salts, and oxidized cellulose such as oxidized regenerated cellulose (ORC) are known for use as wound dressings. Alginate and ORC have a hemostatic effect when applied to wounds.

  European Patent Application No. 0437705 discloses a method of obtaining a neutralized ORC fabric by reacting ORC with a buffer of a salt of a weak acid such as sodium acetate. It is said that the use of a buffer reagent makes it difficult for the ORC cloth to decompose. Neutralizing ORC can support acid sensitive hemostats such as thrombin or t-PA.

  PCT WO 98/00180 discloses the use of collagen lyophilized sponge mixed with oxidized regenerated cellulose (ORC) for the treatment of chronic wounds. This mixed sponge promotes healing of chronic ulcers such as venous ulcers, decubitus ulcers, and diabetic ulcers. This specification states that sulfadiazine silver or chlorhexidine can be included as preservative additives.

  British Patent Application No. 748283 discloses various metal-polysaccharide complexes, including silver. WO 91/11206 discloses the use of silver alginate salts for wound dressings. WO 87/05517 discloses a silver salt of hyaluronic acid that can be used as or included in an antimicrobial wound dressing.

  PCT WO 02/43743 discloses a wound dressing comprising a silver salt of an anionic polysaccharide and a substance that improves the light stability of the silver salt. As this light-stable substance, ammonia, ammonium salt, thiosulfate, chloride, and / or peroxide can be mentioned. In a preferred embodiment, the light stabilizer is ammonium chloride water.

  The present invention provides a wound dressing material comprising a complex of silver and anionic polysaccharide. The material includes about 0.1 wt% to about 3 wt% silver.

As used herein, the term “complex” refers to a homogeneous mixture on a molecular scale, and preferably silver and polysaccharide are ionically or covalently bound. The complex preferably includes a salt formed of an anionic polysaccharide and Ag ⁺ , but may also include a silver cluster and / or colloidal silver metal formed, for example, by exposing the complex to light. it can.

  Preferably, the anionic polysaccharide is a polycarboxylate. Suitable anionic polysaccharides include alginate, hyaluronic acid, pectin, carrageenan, xanthan gum, cellulose derivatives such as dextran sulfate, carboxymethylcellulose, and oxidized cellulose.

  As used herein, the term “oxidized cellulose” refers to any substance produced, for example, by oxidation of cellulose with dinitrogen tetroxide. By such oxidation, the primary alcohol group of the polysaccharide residue is converted to a carboxylic acid group, and a uronic acid residue is formed in the cellulose chain. Since the selectivity of the oxidation reaction is not complete, the hydroxyl groups of carbon 2 and carbon 3 may be converted to keto forms. Such keto units induce alkali labile bonds that cleave the lactone and sugar rings at pH 7 and above to initiate polymer degradation. Thus, oxidized cellulose is biodegradable and bioabsorbable under physiological conditions.

  The oxidized cellulose suitable for use in the present invention is oxidized regenerated cellulose (ORC) produced by oxidation of regenerated cellulose such as rayon. For some time, ORC has been known to have hemostatic properties. ORC has been commercially available since 1950 as a hemostatic fabric called SURGICEL (registered trademark of Johnson & Johnson medical, Inc.). This product is manufactured by oxidizing knitted rayon material.

  The anionic polysaccharide is preferably substantially insoluble in water at pH 7. The molecular weight of the anionic polysaccharide is preferably greater than about 20,000, more preferably greater than about 50,000. The anionic polysaccharide is preferably a fiber having a film shape or length exceeding 1 mm.

  The mass of silver in the complex is preferably about 0.1 wt% to about 50 wt%, more preferably about 1 wt% to about 40 wt%, more preferably 2 wt% to 30 wt%, based on the mass of the anionic polysaccharide, Most preferably from about 5 wt% to about 25 wt%.

  It has been found that silver-containing composites are suitable for the production of antimicrobial wound dressing materials. Silver imparts antibacterial properties to the wound dressing. More surprisingly, the composite silver has the effect of growing cells that heal wounds at low concentrations of about 0.1 wt% to about 3 wt%, and the wound of the present invention can be achieved by applying the composite of the present invention directly to the wound. It is expected to be cured. The trace effects of silver on cultured cells are known to affect both bacterial and cellular growth. Thus, the discovery that the bandage silver of the present invention can have an adverse effect on certain cells important for wound healing is surprising.

  Surprisingly, it has been found that wound dressing materials containing the low levels of silver described above have a surprising ability to suppress the production of TNF-α and IL-1. This is expected to lead to the beneficial anti-inflammatory activity of the wound dressing material.

  The wound dressing material according to the present invention can be in any convenient form such as powder, microspheres, flakes, mats or films.

  In certain embodiments, the wound dressing material according to the present invention is in the form of a semi-solid or gel ointment for topical application.

  In certain embodiments, the wound dressing material according to the present invention is in the form of a lyophilized sponge or a solvent-dried bioabsorbable sponge for application to a chronic wound. The average pore size of the sponge is preferably 10 μm to 500 μm, more preferably 100 μm to 300 μm.

  In another embodiment, the wound dressing material according to the present invention is in the form of a flexible film that can be continuous or intermittent (eg, perforated). The flexible film preferably contains a plasticizer such as glycerol that imparts flexibility.

The wound dressing material is preferably in the form of a sheet such as a sheet having a substantially uniform thickness. The area of such a sheet is typically about 1 cm ² to about 400 cm ² and its thickness is typically about 1 mm to about 10 mm. The sheet can be, for example, a dry frozen sponge, or a net fiber sheet, a woven fiber sheet, a non-woven fiber sheet, or a gel sheet. The sheet preferably contains about 15 wt% water.

  The wound dressing material comprises a silver-containing composite, preferably from about 0.1 wt% to 100 wt%, more preferably from about 0.1 wt% to 5 wt%, such as from about 0.2 wt% to about 2 wt%. The amount of silver in the wound dressing material is about 0.1 wt% to about 3 wt%, preferably about 0.1 wt% to about 1 wt%, such as about 0.2 wt% to about 0.6 wt%, generally about 0 wt%. .3 wt%. If the amount of silver is small, the antibacterial effect is insufficient. If the amount of silver is large, the growth inhibitory effect on cells that heal wounds increases.

  The wound dressing material according to the present invention may further comprise one or more types of polysaccharides that are not complexed with silver. Such polysaccharides can include any one or more types of anionic polysaccharides described above as suitable for complexing with silver. Alternatively or in addition, other polysaccharides include, for example, regenerated cellulose such as cellulose, rayon, non-anionic cellulose derivatives such as hydroxyethyl cellulose, and any other medically acceptable polysaccharide such as starch derivatives. be able to.

  The wound dressing material according to the present invention further comprises an anion complexed with a therapeutic metal ion other than silver, such as, for example, bismuth, copper, nickel, zinc, manganese, magnesium, gold, or combinations thereof. Polysaccharides can be included. The amount of polysaccharide complexed with such other metals is preferably about 0.01 wt% to 10 wt%, more preferably about 0.01 wt% to 1 wt% of the wound dressing material. The amount of such other metals is preferably 10 ppm to 10,000 ppm, more preferably about 50 ppm to about 1,000 ppm of the wound dressing material.

  In addition to polysaccharides, the wound dressing material according to the present invention further comprises textile fibers such as nylon and polyester artificial fibers, non-woven fibers such as melt blown nylon fibers, and bioabsorbable fibers such as polylactide / polyglycolide fibers. Other medically acceptable materials including can be included. Polysaccharides can be reinforced and diluted using such fibers. Such fibers are preferably up to about 90 wt% of the wound dressing material, more preferably from about 25 wt% to about 75 wt%.

  The wound dressing material according to the present invention may further comprise collagen in addition to the silver complex of the anionic polysaccharide. The amount of collagen in the dressing material is preferably about 10 wt% to about 90 wt%, more preferably about 25 wt% to about 75 wt%, based on the dry mass of the wound dressing material.

  Such collagen can be selected from natural collagen such as type I, type II, type III natural collagen, atelopeptide collagen, regenerated collagen, and gelatin.

  In some embodiments, at least a portion of the collagen of the wound dressing material is complexed with silver. This can be achieved by treating the collagen with a silver salt solution. The silver salt can be, for example, silver acetate or silver nitrate at a concentration of about 0.01 mole to about 1 mole. This treatment is preferably carried out at a pH of about 5 to about 9. This silver is thought to be mainly complexed with nitrogen-containing side chains of collagen amino acids, specifically lysine, hydroxylysine, asparagine, glutamine, and arginine. Silver, if present, can also bind to the sulfhydryl groups of methionine and cysteine residues, and the carboxyl groups of aspartic acid and glutamic acid.

  The amount of silver in the collagen complex is preferably about 0.01 wt% to about 30 wt%, more preferably about 0.1 wt% to about 20 wt%, and even more preferably about 2 wt% to about 10 wt%, based on the mass of the collagen. It is. The amount of silver-collagen complex in the wound dressing material is preferably from about 0.1 wt% to about 10 wt%, more preferably from about 0.1 wt% to about 2 wt%. In either case, the total amount of silver in the wound dressing material is generally as described above.

  In certain embodiments, the wound dressing material according to the present invention is fully resorbable. The wound dressing material according to the invention is preferably suitable for direct application to the wound surface.

  Suitable wound dressing materials according to the present invention are disclosed in PCT International Application No. 98/00180 or European Patent Application No. 1153622, the contents of which are hereby incorporated by reference in their entirety. It is a type of bandage material containing silver. In short, such a dressing material is a lyophilized sponge comprising a mixture of collagen and ORC, preferably a lyophilized sponge consisting essentially of such a mixture. The mass ratio of collagen to ORC is preferably about 40:60 to about 60:40. In the sponge according to the invention, a part of this ORC can be replaced with a silver / ORC complex and / or a part of the collagen can be replaced with a silver / collagen complex. Preferably, about 0.1 wt% to about 50 wt% of the ORC is replaced with a silver-ORC complex, and the collagen can be similarly replaced at a similar rate. It should be understood that silver ion exchange can occur between collagen and ORC in such collagen / ORC wound dressing materials.

  A particularly suitable wound dressing material is a lyophilized sponge comprising about 35 wt% to about 60 wt% ORC, about 60 wt% to about 35 wt% collagen, about 0.5 wt% to about 5 wt% ORC / silver complex. . The composite includes about 10 wt% to about 40 wt% silver based on its dry weight. In a preferred embodiment, the material comprises about 0.5 wt% to about 2 wt% ORC / silver composite. In a preferred embodiment, the material consists essentially of collagen, ORC, ORC / silver composite.

  In another aspect, the present invention provides a wound dressing comprising a wound dressing material according to the present invention. This wound dressing consists or consists essentially of a wound dressing material according to the invention.

This wound dressing is preferably in the form of a sheet and comprises an active layer of wound dressing material according to the invention. This active layer is the layer that normally contacts the wound during use, but in some embodiments can be separated from the wound by a liquid permeation prevention sheet. The area of the active layer is preferably about 1 cm ² to about 400 cm ² , more preferably about 4 cm ² to about 100 cm ² .

  The wound dressing preferably further comprises a backing sheet that overlies the active layer opposite the wound side of the active layer. The backing sheet is preferably larger than the active layer and has a width of about 1 mm to 50 mm, preferably 5 mm to 20 mm, extending around the active layer to form a so-called island type bandage. In such a case, it is preferable to coat a pressure sensitive adhesive for medical use on at least the edge portion of the backing sheet.

The backing sheet is preferably substantially liquid impermeable. The backing sheet is preferably semi-permeable. That is, it is preferable that the backing sheet allows water vapor to permeate but does not allow water or exudate to penetrate. The backing sheet is also preferably impermeable to microorganisms. Suitable backing sheet of a continuous and flexible, the temperature of 37.5 ° C., under the conditions of a relative humidity difference of 100% to 10%, preferably 24 hours ^{300g / m 2 ~5,000g / m 2} , more preferably preferably has a moisture vapor transmission rate of ^{50g / m 2 0~2,000g / m 2} (MVTR). The thickness of the backing sheet is preferably in the range of 10 μm to 1,000 μm, more preferably 100 μm to 500 μm.

  The overall MVTR of the bandage according to the present invention is lower than that of the backing sheet alone because the perforated sheet partially prevents the passage of water vapor through the bandage. Preferably, the MVTR of the bandage (measured at the island portion of the bandage) is 20% to 80%, more preferably 20% to 60%, most preferably about 40% of the MVTR with the backing sheet alone. It has been found that such a water vapor transmission rate allows the wound under the bandage to heal under water vapor conditions without the skin surrounding the wound becoming soft.

  Suitable polymers for forming the backing sheet include polyurethanes, polyalkoxyalkyl acrylates, and methacrylates as disclosed in British Patent Application No. 1280631. The backing sheet preferably comprises a continuous layer of high density barrier polyurethane foam, the majority of which are closed cells. A suitable backing sheet material is a polyurethane film sold as ESTANE 5714F®.

The adhesive layer (if included) should be water vapor permeable and / or patterned to allow water vapor to pass through. Adhesive layers are conventionally used in island wound dressings such as acrylate ester copolymer based, polyvinyl ethyl ether based, and polyurethane based pressure sensitive adhesives as described, for example, in UK Patent Application No. 1280631. It is preferably a pressure-sensitive adhesive layer that allows water vapor to permeate continuously. The basic mass of the adhesive layer is preferably 20 to 250 g / m ² , more preferably 50 to 150 g / m ² . A polyurethane-based pressure sensitive adhesive is preferred.

  Furthermore, a multilayer absorbent layer can be formed between the active layer and the protective sheet. For example, such a layer can include a perforated plastic film that supports the active layer in use. In such cases, the pores of the film are preferably aligned with the pores of the hydrogel layer.

The bandage can further include an absorbent layer between the active layer and the protective sheet, particularly when used on an exuding wound. This optional absorbent layer can be any of those conventionally used to absorb wound exudate, serum, or blood in the field of wound healing, including gauze, nonwovens, superabsorbents, hydrogels, and combinations thereof. It can be a layer. The absorbent layer comprises a layer of absorbent foam, such as an open cell hydrophilic polyurethane foam manufactured according to European Patent Application No. 0541391, the contents of which are hereby incorporated by reference in its entirety. preferable. In another embodiment, the absorbent layer may be a nonwoven fiber, such as a viscose artificial carded web. Basis weight of the absorbent ^{layer, 50g / m 2 ~500g / m} 2 range, can be, for example, ^{100g / m 2 ~400g / m 2} . The thickness of the uncompressed absorbent layer may be in the range of 0.5 mm to 10 mm, for example, 1 mm to 4 mm. The open (non-compressed) liquid absorption value measured with saline can be in the range of 5-30 g / g at 25 ° C. The absorbent layer preferably extends substantially similar to the chitosan / ORC layer.

  The side of the dressing facing the wound is preferably protected with a removable cover sheet. This cover sheet is usually formed from a flexible thermoplastic material. Suitable materials include polyesters and polyolefins. The adhesive surface of the cover sheet is preferably a release surface. That is, the release surface is weakly adhered to the adhesive of the active layer and the backing sheet, and makes it easy to peel the cover sheet from the hydrogel layer. For example, the cover sheet can be formed from a non-adhesive plastic such as a fluoropolymer or can be provided with a release coating such as silicon or a fluoropolymer release coating.

The wound dressing material and / or wound dressing according to the present invention is preferably sterilized. They are also preferably packaged in a container through which microorganisms cannot pass. The sterility assurance level is preferably higher than 10 ⁻⁶ . The bandage is preferably sterilized with gamma rays.

  In a further aspect, the present invention provides a complex of silver and anionic polysaccharide for the production of a wound dressing material according to the present invention for the treatment of wounds, especially chronic wounds such as venous ulcers, pressure ulcers or diabetic ulcers. Including using. Preferably, the treatment includes applying the wound dressing material directly to the wound surface.

  In a further aspect, the present invention provides a method of treating a wound. The method includes applying a wound dressing material comprising an effective amount of an anionic polysaccharide and silver complex to the wound. Although this complex has an antibacterial effect, it does not have significant antiproliferative activity on cells that heal wounds. The wound dressing material preferably has the effect of suppressing inflammation. This method is particularly suitable for the treatment of chronic wounds such as venous, pressure ulcers, or diabetic ulcers.

  The material according to the present invention has surprisingly been found to be effective in inhibiting the release of TNF-α from macrophages. This property is expected to make the material effective for the treatment of inflammation. Accordingly, in another aspect, the present invention includes using an anionic polysaccharide and silver complex in the manufacture of a material comprising about 0.1 wt% to about 3 wt% silver for use in treating inflammation.

  The complex of anionic polysaccharide and silver contained in the material of the present invention can be obtained by a method including a step of treating the anionic polysaccharide with a silver salt solution. This solution is preferably an aqueous solution.

  The anionic polysaccharide is preferably substantially insoluble in pH 7 water. Accordingly, the solid polysaccharide is treated. For example, the polysaccharide can be in the form of solid fibers, sheets, sponges, or fabrics. In certain embodiments, the anionic polysaccharide is a salt, and thus the treatment can be considered an ion exchange. In another embodiment, the anionic polysaccharide is at least partially in the free acid form. In such a case, the silver salt is preferably a salt of a weak acid such as silver acetate. Thus, the anionic polysaccharide is at least partially neutralized by the silver salt. A similar process is disclosed in European Patent Application No. 0437705, which is hereby incorporated by reference in its entirety.

  The neutralization reaction can be carried out with only water or alcohol, but is preferably carried out with a mixture of water and alcohol. By using a mixed solution of water and alcohol, the solubility of the weak acid salt is increased by water, and excessive swelling, distortion, and weakening of the anionic polysaccharide during the neutralization reaction are prevented by the alcohol. Therefore, the physical properties of the material are maintained. Methanol is a suitable alcohol because many of the above-mentioned salts dissolve well in a mixture of ethanol and water. The ratio of alcohol to water is preferably in the range of about 4: 1 to 1: 4. If there is too much alcohol in the mixed solution, particularly when the alcohol is other than methanol, some salts cannot be dissolved. If the amount of water in the mixed solution becomes too large, the polysaccharide will expand during the neutralization reaction, and some physical properties such as the tensile strength of the polysaccharide will be lost. Other useful alcohols can include, for example, ethyl alcohol, propyl alcohol, and isopropyl alcohol.

  A mild neutralizing agent such as silver acetate can be used to control the degree of neutralization. Even if a stoichiometrically and chemically equivalent amount of neutralizing agent and anionic polysaccharide carboxylic acid are used, a 100% neutralized polysaccharide cannot be produced. This is because it is produced by a strong irreversible reaction with a base such as sodium hydroxide, sodium carbonate, sodium hydrogen carbonate and ammonium hydroxide.

  The anionic polysaccharide acts as an ion exchanger and removes from the solution any silver salt silver cations that pass through it. The by-product of this exchange is an acid from the salt, and the use of a weak organic acid salt produces a weak acid such as acetic acid that does not destroy the polysaccharide. When a salt of a strong acid such as sodium chloride or sodium sulfate is used, hydrochloric acid or sulfuric acid is generated as a by-product, and these strong salts cause depolymerization of the polysaccharide, thereby destroying the polysaccharide.

  When a weak acid silver salt is used, silver ions are exchanged for polysaccharide protons, and a portion of the salt is converted to a weak acid. By mixing the acid and salt in such a solution, a buffer solution is obtained, the pH is maintained almost constant, and the degree of neutralization is controlled. An equilibrium reaction is established and silver ions bind to the acid portion of the polysaccharide and the salt molecule. Such partitioning of silver ions does not terminate the neutralization reaction of the polysaccharide.

  For example, the theoretical amount of silver acetate can be used to neutralize the carboxylic acid groups of the oxidized cellulose polymer by about 65% to 75%. By adjusting this pH by self-buffer generation and controlling the expansion of the material by using methanol, the material is partially neutralized and physical properties such as the tensile strength and shape of the polysaccharide are maintained.

  The amount of silver salt to be used is usually about the same amount as the theoretical amount of carboxylic acid in the polysaccharide, and up to twice as much. Alternatively, a second charge of the theoretical amount of silver salt can be used when the reaction is recharged with fresh solvent and salt after the first charge has reached a certain pH. The elevated pH material is then washed to remove excess silver salts and ions.

  It should be understood that the composite obtained by the above method can be used in products and methods according to any aspect of the present invention. More generally, any feature of the invention described as preferred or all features described using the methods described above, or combinations thereof, are preferred in all other embodiments of the invention. Furthermore, any combination of particular features or preferred features described herein are also included in the scope of the disclosure.

  Particular embodiments of processes and products according to the present invention will now be described by way of example only with reference to the accompanying drawings.

Example 1
A composite of ORC and silver was prepared as follows.

  Using a rotary knife cutter in the screen plate, a SURGICEL fabric (Johnson & Johnson Medical, Arlington, Arlington) was crushed into fibrous ORC powder while maintaining below 60 ° C. Prepared.

  Silver acetate powder (4.08 g) was dissolved in 800 ml of deionized water. When all the powder dissolved to a clear colorless liquid, ground ORC fiber (5 g) was added to this solution. The ORC was then allowed to react for 60 minutes or less if desired. The solution was then filtered to collect the fiber and washed with deionized water or alcohol. The ORC silver composite was then dried at 37 ° C. overnight or until the fibers were dry. In order to reduce the darkness of the final product, the reaction solution and the reacted fibers were not exposed to light during the reaction.

Example 2-7
A lyophilized collagen / ORC sponge suitable for use as a wound dressing material according to the present invention was prepared as follows.

  First, a collagen component was prepared from bovine dermis as follows. The bovine dermis is separated from the skin, scraped and immersed in a sodium hypochlorite solution (0.03% w / v) to prevent microbial activity from interfering with subsequent processing.

  The dermis is then washed with water and treated with a solution containing sodium hydroxide (0.2% w / v) and hydrogen peroxide (0.02% w / v) at ambient temperature to expand the dermis. Sterilized.

  The dermal strip is then loaded with sodium hydroxide, calcium hydroxide, and sodium bicarbonate (0.4% w / v, 0.6% w / v, and 0.05% w / v, respectively). The containing solution was tumbled until the amide nitrogen level was lower than 0.24 mM / g over a period of pH 12.2, ambient temperature, 10-14 days, and an alkali treatment step was performed.

  The dermal strip was then subjected to an acid treatment step at ambient temperature, pH 0.8-1.2, 1% hydrochloric acid. This treatment continued to tumbl until the dermis strips fully absorbed the acid and the pH was below 2.5. The strip was then washed with water until the pH was 3.0-3.4.

  The dermis strips were then crushed with ice in a ball chopper, initially with a coarse setting and then with a fine setting. The resulting paste of 650 g dermis strips per 100 g water as ice was frozen and stored until use in the next step. However, the collagen was stored without lyophilization until it was mixed with ORC in the next step.

Then in a sufficient amount of water acidified with acetic acid to a pH value of 3.0, silver-ORC complex, ground unmodified ORC powder, the required amount (depending on the amount of solids) of frozen collagen paste, It added so that it might become the mass ratio shown below and total solid content might be 1.0%-2.0%.
Example 2: 45% Silver-ORC / 55% Collagen Example 3: 20% Silver-ORC + 15% ORC / 55% Collagen Example 4: 10% Silver-ORC + 35% ORC / 55% Collagen Example 5: 2% Silver-ORC + 43% ORC / 55% Collagen Example 6: 1% Silver-ORC + 44% ORC / 55% Collagen Example 7: 0.2% Silver-ORC + 44.8% ORC / 55% Collagen

  The mixture was homogenized using a Flyma MZ130D homogenizer with gradually finer settings to a homogeneous slurry. The pH of this slurry was maintained at 2.9-3.1. The temperature of the slurry was maintained below 20 ° C. and the solid content was maintained at 1% ± 0.07.

  The resulting slurry was transferred to a deaerator. A 30 minute evacuation was started with intermittent stirring so that the gas in the slurry escaped. The slurry was then transferred to a freeze drying tray to a depth of 25 mm. The tray was placed on a freezer shelf set at -40 ° C. The freeze drying program was then started and dried, and collagen and ORC were cross-linked by dehydration freezing to form a thick sponge pad.

  When this cycle was complete, the vacuum was released, the lyophilized block was removed, the top and bottom layers were removed, and the block was then divided into 3 mm thick pads. The step of dividing the lyophilized block into pads was performed with a Fecken Kirfel K1 slitter.

  Finally, these pads were cut to the desired size and shape with a die cutter, packaged and sterilized with 18-29 KGy cobalt 60 gamma radiation. Surprisingly, this irradiation did not cause significant denaturation of the collagen. This is considered as stabilization due to the presence of ORC. The resulting freeze-dried collagen ORC pad had a uniform white velvety appearance.

Example 8-11
A collagen / ORC sponge in which a portion of collagen was replaced with a collagen-silver complex as shown below was prepared from unmodified ORC and collagen.

  Silver acetate powder (1.48 g) was placed in 400 ml of deionized water and dissolved by stirring and warming.

  Collagen slurry (417 g, solid is 5 g at 1.2% of the slurry) was added to the silver acetate solution and allowed to react for 10 minutes with slow stirring (collagen became fibrous when reacted for a longer time) It becomes difficult to re-slurry with acetic acid).

  The solution was filtered using a funnel and filter paper and the solid was washed with deionized water. The filter paper was gently pressed from both sides to squeeze out excess liquid contained in the solid. In this step and subsequent steps, the collagen solids were prevented from being exposed to light.

  This solid was brought to a mass of 417 g with 0.05 M acetic acid. This mixture was placed in an industrial Waring Blender and mixed until a smooth slurry was obtained.

A lyophilized sponge was then made using unmodified ORC and a collagen slurry partially substituted with a silver-collagen composite slurry as described in Example 2-7. These examples are considered to be within the scope of the present invention. Because of the silver-collagen complex silver also forms and exchanges with the ORC under the preparation conditions. The following mixtures were prepared:
Example 8: 55% silver-collagen / 45% ORC
Example 9: 45% silver-collagen + 10% collagen / 45% ORC
Example 10: 30% silver-collagen + 25% collagen / 45% ORC
Example 11: 15% silver-collagen + 40% collagen / 45% ORC

Step 1
The bactericidal activity of the sponge prepared in Example 2-7 against Pseudomonas Aeruginosa and Staphylococcus Aureus was examined from the inhibition zone.

  Six squares (2 cm x 2 cm) were cut from each sample under sterile conditions. On the first day of the experiment, both P. aeruginosa and S. aureus cultures were incubated aerobically at 37 ° C. for 24 hours in Diagnostic Sensitive Agar (DSA). After 24 hours, each test sample was placed in a DSA plate and quickly wetted with 0.5 ml buffer. Three square samples were placed on the plate and inoculated with P. aeruginosa, and another three samples were placed on the plate and inoculated with S. aureus. The plates were then incubated for 24 hours at 37 ° C. The area around the sample where growth was blocked was then measured with a caliper and the test sample was placed in a freshly inoculated DSA plate. The lower part of the sample was swab tested. DSA plate swabs were applied and incubated for 24 hours and examined for growth to determine if the sample was bacteriostatic, if not sterilized. The sample was transferred to a freshly inoculated plate and the procedure described above was performed every 24 hours for 72 hours as long as the sample was intact.

  As a negative control, a 45% ORC / 55% collagen lyophilized sponge without any silver was tested. A commercially available silver-containing antibacterial bandage (ACTICOAT, a registered trademark of Smith & Nephew) and silver nitrate solution (0.5%) were used as positive controls, and both inhibition bands were observed during the test. .

  Referring to FIGS. 1 and 2, it can be seen that a material containing 1% or more of silver-ORC has a significant bactericidal effect against Staphylococcus aureus. This ability corresponds to that of ACTICOAT® bandage.

  Referring to FIG. 3, it can be seen that the material containing 10% or more of silver-ORC had a significant bactericidal effect against Pseudomonas aeruginosa. The ability after 24 hours was superior to that of ACTICOAT® dressing.

  The bactericidal activity of the sponge prepared in Example 8-11 was tested by examining the inhibition zone in the same manner as described above for Pseudomonas aeruginosa. Again, 55% collagen / 45% ORC sponge was used as a negative control and silver nitrate solution (0.5%) and ACTICOAT® were used as positive controls. The results after 48 hours are shown in FIG. It can be seen that the sponge made with the collagen-silver complex shows a positive control and an inhibition band similar to the silver-ORC complex of FIG.

Step 2
The growth inhibitory effect of the dressing material produced in the above example was evaluated as follows.

  Prototype extracts were prepared by placing 1 mg of each wound dressing material to be tested in 1 ml of serum-free medium and incubating at 37 ° C. for 24 hours under sterile conditions.

Adult human dermal fibroblasts were grown and maintained in DMEM 10% FBS (standard medium, Dulbecco's minimal essential medium, fetal bovine serum). These cells were subcultured and used for experiments at 95% confluence. Adult human dermal fibroblasts were harvested at 95% confluency and seeded again in DMEM + 10% FBS in 96-well microtiter plates (100 μl / well) at a cell density of 2.5 × 10 ⁴ cells / ml. The cells were incubated for 24 hours in a humidified incubator (37 ° C., 5% CO ₂ ) so that the cells could adhere to the plate surface. The medium was then aspirated and the cell monolayer was washed with serum-free DMEM. Test samples (extracts of each prototype) were added to a monolayer of cells (100 μl / well) and tested 6 times at each concentration. Serum-containing growth medium (DMEM 10% FBS) was used as a positive control, and serum-free medium was used as a negative control. All samples were incubated with cells for 48 hours at 37 ° C., 5% CO ₂ . After this incubation, the conditioned medium was removed by aspiration and replaced with a labeling solution from Cell Proliferation Kit II (Catalog No. 1465015) available from Boehringer Mannheim. Once this labeling solution was added, the initial absorbance at 450 nm was measured, then the microtiter plate was incubated at 37 ° C., 5% CO ₂ and the absorbance was monitored for 4 hours. The proliferation effect of each prototype was evaluated by comparing the absorbance measured against the positive and negative controls.

  Since 45% ORC / 55% collagen lyophilized sponge without any silver was found to promote fibroblast proliferation, it was tested as a positive control.

  ACTICOAT® was used as a negative control because it was known that silver's effect on cultured cells was toxic and caused cell death at high concentrations.

  The result shown in FIG. 4 is very surprising. Samples containing 0.2% silver-ORC complex and 1% silver-ORC complex were found to significantly promote fibroblast growth, respectively. This effect was at least twice that observed with collagen / ORC alone, which was already known to stimulate cell proliferation. This stimulating effect was limited to low concentrations of silver-ORC and was detrimental to cell growth at concentrations of 2% and above. The ACTICOAT® control of this experiment showed the expected adverse effects on fibroblast growth.

  Referring to FIG. 6, a sample of collagen / ORC material (Example 9) made with 45% silver-collagen, 10% unmodified collagen, and 45% ORC stimulates fibroblast proliferation, but collagen It can be seen that it does not stimulate as much as / ORC positive control.

Step 3
The possible anti-inflammatory activity of the wound dressing material according to the present invention was investigated as follows.

  Lipopolysaccharide (LPS) has been shown to induce the production of TNF-α and IL-1 in monocyte macrophage cells. Since TNF-α and IL-1 have been shown to promote inflammatory processes, this response can be used to assess the potential anti-inflammatory activity of wound dressings.

  To assess the anti-inflammatory activity of the wound dressing material, the samples were monocytes (THP-1) in the presence and absence of lipopolysaccharide (E.Coli 055.B5, Sigma Chemical Co.). Incubated with cells, obtained from the European Collection of Cell Cultures. As positive and negative controls, various reference dressing materials and various concentrations of silver acetate were incubated with THP-1 cells in the presence and absence of LPS. The concentration of silver acetate was in the range of 0.01M to 0.001 mM. Silver acetate is known to be cytotoxic at high concentrations in this range and not cytotoxic at low concentrations. The levels of both TNF-α and IL-1 produced by THP-1 cells were measured for 24 hours after addition of LPS.

The following wound dressing materials were tested.
(A) Lyophilized sponge wound dressing material according to the present invention comprising 1 wt% silver / ORC composite prepared as described in Example 6 (B) Johnson & Johnson Wound Care Freeze-dried sponge (C) Johnson & Johnson made of 55 wt% collagen and 45 wt% ORC, prepared as PROMOGRAN (R) and prepared as disclosed in European Patent Application No. 1153622 Medical gauze sold as SOF-WICK (R) by Johnson & Johnson Wound Care

  The experimental procedure is as follows.

THP-1 cells were left in low concentration LPS 10% FBS. These cells were then spun down at 1,000 rpm for 10 minutes and resuspended to a concentration of 10,000 cells / ml. PMA (Phorbol 12-myristate 13-acetate, Sigma Chemical Co.) was then added to the cell suspension to a final concentration of 2.4 × 10 ⁻⁷ M.

This cell suspension was then used to seed in 24-well microtiter plates at 10,000 cells / well. The plate was then incubated for 72 hours at 36 ° C., 5% CO ₂ to allow the cells to adhere to the tissue culture plastic.

  RPMI medium containing 1 mg / ml LPS was prepared (LPS was returned to 1 mg / ml with sterile phosphate buffered saline). After incubation, the 24-well microtiter plate PMA medium was aspirated and replaced with RPMI + LPS or RPMI medium.

  A sample punched to 6 mm from the material to be tested was prepared and immersed in PBS for 2 minutes or less. Two samples from each type of dressing material were sampled four times, giving a total of 8 samples for each type of dressing material. Silver acetate solutions were prepared in RPMI medium to concentrations of 0.1 M, 10 mM, 1 mM, and 0.1 mM.

  Samples of pre-soaked dressing material and different concentrations of silver acetate were added to the microtiter plate. 10 μl of each silver acetate solution was added to each test well to give test concentrations of 0.01 M, 1 mM, 0.1 mM, and 0.01 mM.

The microtiter plate was then incubated at 37 ° C., 5% CO ₂ . After 4 hours, 6 hours, and 24 hours of incubation, 20 μl aliquots of conditioned medium were removed from each well. Samples were transferred to bullet tubes and stored at -70 ° C until ELISA analysis.

  Prior to ELISA analysis, media samples were divided into 10 μl aliquots and diluted 1: 2 with calibration diluent (the calibration dilution used depends on the ELISA being performed). This dilution brought the sample within the standard curve range. The manufacturing instructions for the ELISA kit were followed. The ELISA kit was obtained from R & D Systems Limited.

  Standard curves for both TNF-α and IL-1β were plotted and the absorbance of the sample was converted to the corresponding cytokine concentration using the derived formula. Average concentrations of both TNF-α and IL-1β were calculated for each sample condition at each time. These results were then used to compare differences in test conditions over time.

  At the cytotoxic level (0.01M), TNF-α was not produced because silver acetate induced cell death. At weak cytotoxin levels (0.1 mM and 0.001 mM), THP-1 cells survived (confirmed by microscopic examination) and a dose response curve of the produced TNF-α could be obtained. 0.01 mM silver acetate has the ability to suppress the amount of TNF-α produced, but the lowest concentration of silver acetate does not affect the amount of TNF-α produced after stimulation with LPS I understood.

  Referring to FIG. 7, it can be seen that the various bandage materials tested differ in their ability to affect the amount of TNF-α produced by THP-1 cells. Gauze (Sample C) had little effect and cells present in this bandage produced the same level of TNF-α as the positive control (LPS only).

  Cells present in the silver-free collagen / ORC sponge bandage (Sample B) produced lower levels of inflammatory cytokines than LPS alone.

  However, with silver-containing collagen / ORC sponge dressing material according to the present invention (sample A) placed in assay wells, THP-1 cells produced virtually no TNF-α. The inhibitory effect with the material according to the invention was surprisingly superior to the inhibitory effect obtained with a silver acetate concentration (0.01 mM) equal to that present in this material.

  The IL-1β results (data not shown) were similar to the TNF-α results.

  These results show the ability of the material according to the present invention to suppress the production of important inflammatory cytokines by THP-1 monocyte macrophages. This effect has been shown to persist over a 24 hour test period, suggesting that the material of the present invention is capable of suppressing cytokine production over an extended period of time. The anti-inflammatory activity of the material according to the invention could not be inferred from only two components.

  The examples presented here are for illustrative purposes only, and many other configurations and methods within the scope of the present invention will be apparent to those of ordinary skill in the art who have read this specification.

It is the graph which plotted the antibacterial effect with respect to S. aureus (Staphylococcus Aureus) 2 days after in the material of the 1st range of wound dressing material as a stop zone (unit: mm). It is the graph which plotted the antibacterial effect with respect to Staphylococcus aureus after 24 hours and 48 hours in the material of the 2nd range of wound dressing materials as a stop zone (unit: mm). It is the graph which plotted the antibacterial effect with respect to Pseudomonas Aeruginosa after 24 hours, 48 hours, and 72 hours in the material of the 2nd range of wound dressing material as a zone (unit: mm). . FIG. 5 is a graph showing the effect on cell growth of an extract from a third range of materials of wound dressing material. It is the graph which plotted the antibacterial effect with respect to Pseudomonas Aeruginosa after 2 days in the material of the 4th range of wound dressing material as a stop zone (unit: mm). FIG. 6 is a graph showing the effect on cell growth of an extract from a fifth range of materials of wound dressing material. (A) wound dressing material according to the invention, (B) 0.01M silver acetate solution, (C) lyophilized collagen / ORC dressing without silver, and (D) incubated with lipopolysaccharide and monocyte macrophages It is a graph figure of TNF- (alpha) density | concentration with respect to time of the conventional wound dressing gauze.

Claims (15)

  A wound dressing material comprising a complex of anionic polysaccharide and silver, wherein the material comprises silver in the range of about 0.1 wt% to about 3 wt%. The wound dressing material according to claim 1, wherein the complex includes a salt formed with the anionic polysaccharide and Ag ⁺ .   The wound dressing material according to claim 1 or 2, wherein the anionic polysaccharide is a polycarboxylate.   4. The anionic polysaccharide is selected from the group consisting of alginate, hyaluronic acid, pectin, carrageenan, xanthan gum, dextran sulfate, cellulose derivatives, oxidized cellulose, and combinations thereof. Wound dressing material.   The wound dressing material according to claims 1 to 4, wherein the amount of silver in the complex is about 1 wt% to about 50 wt% based on the mass of the anionic polysaccharide.   The wound dressing material according to claim 6, characterized in that the material is in the form of a freeze-dried sponge sheet, a woven sheet, a nonwoven sheet or a gel sheet.   7. A wound dressing material according to claim 1-6, wherein the wound dressing material comprises the composite in the range of about 0.1 wt% to about 10 wt%.   8. A wound dressing material according to claim 1-7, wherein the amount of silver in the wound dressing material ranges from about 0.1 wt% to about 2 wt%.   The wound dressing material according to claim 1, wherein the wound dressing material comprises oxidized regenerated cellulose (ORC) and collagen complexed with silver.   A wound dressing comprising the wound dressing material according to claim 1.   The wound dressing according to claim 10, wherein the wound dressing is packaged in a container that is sterile and impervious to microorganisms.   Using an anionic polysaccharide and silver complex in the preparation of a material for use in the treatment of wounds containing silver in the range of about 0.1 wt% to about 3 wt%.   14. Use according to claim 13, characterized in that the wound is a chronic wound, such as a venous ulcer, a pressure ulcer or a diabetic ulcer.   14. Use according to claim 12 or 13, wherein the treatment comprises applying the wound dressing material directly to the surface of the wound. Using an anionic polysaccharide and silver complex in the preparation of a material for use in the treatment of inflammation comprising silver in the range of about 0.1 wt% to about 3 wt%.

Images