JP2645098B2 - Antibacterial agent-containing artificial skin and method for producing the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel artificial skin containing an antibacterial agent and a method for producing the same.

The artificial skin of the present invention is applied to a damaged surface of a skin when the skin is damaged by a wound, a burn, etc., and protects the wound and allows cells having a tissue repair function to enter into the wound, thereby healing the wound. Is to promote.

[Conventional technology and its problems] Temporarily applied to an affected area for the purpose of protecting the affected area due to diseases or wounds such as burns, skin wounds and traumatic skin defect wounds, pressure ulcers, and promoting healing As a wound dressing,
Conventionally, gauze, absorbent cotton, etc. have been used, but this has low antibacterial properties, and the wound surface dries out to quickly absorb exudate, causing pain and bleeding when removing it. there were. In addition, an ointment or the like is also used in combination, but in this case, the absorption of the exudate is insufficient and the wound surface is excessively wet.

Further, as an alternative to these, in particular, when applied to a wide range of wound surfaces, silicone gauze, silicone rubber and nylon having a velor-like surface structure, synthetic materials such as synthetic fiber sheets such as Teflon, etc. Coating films and coating films of biological materials such as freeze-dried pork skin, chitin nonwoven fabric, collagen film, polyamino acid sponge, and mucopolysaccharide composite collagen film are also known. However, among these, the artificial coating film leaves various problems in terms of adhesion to the affected part, water vapor permeability, cracks, and the like, while the coating film of a biological material has characteristics such as biocompatibility. However, many of them have antigenicity, have drawbacks such as bacterial infection and deterioration due to exudate, and have had problems that raw materials are difficult to obtain. More recently, composite membranes consisting of a collagen-treated nylon mesh and a silicone membrane have been developed and put to practical use.They adhere well to the wound surface and have an appropriate moisture permeability, but they adhere to the wound surface and the granulation tissue is covered. There was a drawback that it penetrated into the coating.

In addition, widespread or third-degree burns were more susceptible to infection, causing sepsis and bacterial shock, and eventually life threatening. Long-term administration of antibiotics as a countermeasure against infection resulted in drug resistance of the bacterium, resulting in bacterial replacement, resulting in gram-negative bacilli and fungi. On the other hand, many topical treatments focus on antibacterial properties and permeability, with particular emphasis on gram-negative bacilli such as Pseudomonas aeruginosa. The conditions required as a topical treatment agent for burns are 1) strong antibacterial properties, 2) strong penetrating action that can act on bacteria under the burn surface eschar, 3) low tissue toxicity, 4) analgesic action, 5) promotion of epidermal formation. And promote granulation (Shimazaki Shuji, Trauma Surgery p481, Dentistry Publishing, 1973). Unfortunately, there are currently no topical treatments that meet all of these requirements. At present, silver sulfadiazine, which is an antibacterial agent used, has excellent antibacterial properties against Pseudomonas aeruginosa, and is commercially available in the form of a cream containing 1% silver sulfadiazine. However, because it is a cream base, it has soaked into the gauze bandage together with the exudate, leaving only about 21% of the wound surface (Takehiko Ohura,
Emergency Medicine, 5 1633 (1981)).

As an alternative, a wound dressing made of animal tissue containing silver sulfadiazine (Japanese Patent Publication No. 59-20356)
3) and polyamino acid sponges containing silver sulfazine have been reported recently (Norimitsu Kuroyanagi et al., Journal of the Nikkei Association, 7 526).
(1987)).

On the other hand, as a wound covering film, freeze-dried pig skin, a collagen film, a mucopolysaccharide composite collagen film, a composite film composed of a collagen-treated nylon mesh and a silicone film, and the like are known. At present, satisfactory wound covering films and artificial skins have been obtained, which have disadvantages such as bacterial infection and deterioration due to exudate.

[Problems to be Solved by the Invention] As described above, the conventional wound dressings each have their own drawbacks. Therefore, as a treatment for an affected part when skin tissue is lost due to a burn or the like, autologous transplantation is currently used. It is the best way. However, it is very difficult when the skin defect is extensive, and it is necessary to repeat the transplant several times over a long period of time, even if applicable.

Therefore, it is desired to develop a wound dressing that temporarily or permanently covers the diseased part in place of autologous transplantation to prevent bacterial infection and outflow of body fluids, and to increase tissue cells to promote tissue repair. ing.

Infection is likely to occur in widespread burns and third-degree burns, so infections are prevented by using a cream base containing an antibacterial agent. However, of these, about 57% permeate the gauze bandage with the exudate and reach only about 21% at the wound surface. In addition, in the case of a cream base, the operation surface is inconvenient, such as being applied to the wound surface every day. If the sustained release of the antimicrobial agent can be sustained for a certain period of time, there is no need to expose the wound surface to the outside air, nor to apply it daily. As a wound covering film, the moisture permeation control layer can not only control moisture permeation but also prevent external infection. Furthermore, if an antimicrobial agent is applied to this membrane and it is possible to release it gradually,
It can be applied not only to prevention of infection from the outside but also to cases where the wound surface is contaminated with bacteria.

[Means for Solving the Problems] The above object is achieved by an artificial skin having the following constitution and a method for producing the same.

1) The content of fibrillar collagen and helix is 0 to 80
% Of a wound contact layer composed of a matrix with denatured collagen, a support layer composed of a fibrous collagen matrix having a cross-linked structure, and a moisture permeation control layer, at least one of which is provided with an antimicrobial agent. Artificial skin containing.

2) The artificial skin according to item 1, wherein the antibacterial agent is silver sulfadiazine.

3) The artificial skin according to item 1, wherein the antibacterial agent is gentamicin.

4) Fibrous collagen and helix content 0-80%
Is placed in a container, a fibrous collagen matrix having a crosslinked structure is placed thereon, and lyophilized to form a porous body, and the upper surface of the fibrous collagen matrix is placed on a substrate. Resting on the stretched still sticky moisture permeable material thin film, drying until the thin film is hardened, and finally heat-treating under vacuum less than 0.05 Torr at 50-180 ° C for 1-24 hours Consisting of a mixed solution of fibrillar collagen and denatured collagen,
A method for producing artificial skin, comprising adding an antibacterial agent to at least one layer of a fibrous collagen matrix or a water-permeable substance.

5) The method for producing artificial skin according to item 4, wherein the fibrous collagen matrix is crosslinked by thermal dehydration or chemical treatment.

6) Thermal dehydration treatment is 50 ° C or more under a vacuum of 0.05 Torr or less.
Item 5. The method for producing artificial skin according to item 5, wherein the artificial skin is heated at a temperature of 180 ° C for 1 to 24 hours.

7) The method for producing artificial skin according to item 5, wherein the chemical treatment is a treatment with aldehyde, carbodiimide, isocyanate or epoxy.

As described above, the artificial skin of the present invention comprises three layers: a wound contact layer (lower layer), a support layer (middle layer), and a moisture permeation control layer (upper layer), and at least one of the layers contains an antibacterial agent. I have.

The wound contact layer directly covers and protects the wound surface, softens pain, provides adequate moisture and prevents bacterial contamination. In addition, the wound contact layer spreads very early when macrophages, neutrophils and other inflammatory cells are infiltrated when applied to the wound surface, then diffusely infiltrate fibroblasts and the capillary system early, resulting in the dermis A similar connective tissue is constructed to promote wound healing. Immediately above the wound contact layer, the surrounding healthy epidermis extends and proliferates, and finally, the supporting layer and the moisture permeation control layer thereon are demarcated and eliminated. As a material constituting the wound contact layer, a matrix of fibrillar collagen and denatured collagen having a helix content of 0 to 80% is preferable.

The present invention is particularly effective against infection from the outside, in which the outermost layer contains a moisture permeation controlling layer containing an antibacterial agent, and the water permeation controlling layer is in close contact with the collagenase-resistant and stable fibrosis in vivo. A site which can be in contact with the collagen matrix and the wound consists of a collagen-denatured collagen matrix layer having good cell penetration. However, when the amount of the antibacterial agent is large, it is not possible to form a moisture permeation control layer of silicone, or the physical properties of the silicone film are significantly reduced. Therefore, when the wound surface is contaminated with bacteria or the like and a large amount of the antibacterial agent is required, it is desirable to contain the antibacterial agent in any one of the collagen matrix layers.

The present invention also shows an artificial skin in which a collagen-denatured collagen matrix layer having good cell invasiveness has a collagen as a skeleton and denatured collagen is bonded by thermal dehydration crosslinking. Denatured collagen has a molecular triple-helix impregnation of 0% to 80%,
Denaturation is performed by heat treatment, chemical treatment, physical treatment, etc., and heat denaturation treatment is most preferred. The thermal dehydration crosslinking is performed under vacuum at a temperature of 50 to 180 ° C. at a temperature of less than 0.05 Torr and for a time of 1 hour to 24 hours, preferably at a temperature of 100 to 120 ° C. at a vacuum of less than 0.05 Torr and a time of 2 to 24 hours. The time is not less than 8 hours.

In the present invention, the antibacterial agent may be selected from silver sulfadiazine, gentamicin, silver nitrate and the like, but is not limited to these antibacterial agents.

Hereinafter, the present invention will be described in more detail based on embodiments. FIG. 1 is an enlarged sectional view showing a microstructure of an embodiment of the artificial skin of the present invention. As shown in FIG. 1, the artificial skin 1 of the present invention has a collagen-denatured collagen matrix layer 2 in which the site in contact with the wound portion has good cell invasiveness, and is adjacent to the wound portion contact layer 2 to have collagenase resistance. The collagen matrix layer 3 and the outermost layer, which are stable in the living body, comprise the silicone film layer 4, and the antibacterial agent 5 is contained in at least one of the support layers 2, 3 and 4.

Here, the wound part contact layer 2 having cell invasiveness means that when the artificial skin 1 is applied to a wound part, macrophages, neutrophils and other inflammatory cells are less wet, and Fibroblasts invade, resulting in remodeling of the dermis-like connective tissue. The denatured collagen used in the wound contact layer 2 is subjected to heat treatment, chemical treatment, physical treatment, or the like, but heat treatment is most preferable. The degree of collagen denaturation is indicated by the content of the helical structure. The helix content refers to the triple-stranded helix content unique to collagen. In collagen, since the helix is randomly coiled, the helix content corresponds to the degree of denaturation. The helical content can be measured by circular dichroism or infrared spectrophotometry (PLGordon, IVY
annas, et al, Macromolecules, 7 (6) 954 (1974), Nakura, Hashimoto et al., Polymer Papers 41 (8) 473 (1984)).
The index of the degree of denaturation of collagen used here, that is, the helix content is 0 to 80%, more preferably 0 to 50%.
It is. For example, when a collagen solution is heat-treated at 60 ° C. for 30 minutes, the helix content is about 40%. When heat treatment was performed at 100 ° C. for 24 hours, the helix content became 0, and it was found that a part of the collagen molecule was cleaved by electrophoresis. The composition of the denatured collagen is 5 to 80%, more preferably 10 to 50%.

On the other hand, even if the support layer 2 absorbs exudate and decomposes, the collagen matrix layer 3 having collagenase resistance
It has been found that the shape of the support layer 3 can be maintained. It is desirable that the collagen of the support layer 3 is cross-linked, so that the cells can enter the living tissue for a certain period of time before being replaced with a living tissue.
Essential for maintaining physical properties that continue to provide a growth scaffold. To introduce the high degree of crosslinking required for this, thermal dehydration crosslinking requires exposure to 110 ° C. under vacuum for 24 hours. In the chemical crosslinking, aldehyde-based crosslinking, carbodiimide-based crosslinking, isocyanate-based crosslinking, epoxy-based crosslinking and the like can be selected, but the crosslinking agent is not limited to these. It is desired to crosslink at a crosslinking agent concentration of 0.01% or more, respectively. The collagen to be cross-linked is preferably atelocollagen in which the antigenic determinant has been removed by an enzyme from the aspect of suppressing the expression of antigenicity, but if atelocollagen is used as it is in a dispersed state, the physical properties are not significantly improved even if cross-linking is performed Therefore, it is more preferable that the atelocollagen is subjected to a neutralization treatment (using a phosphate buffer) at 37 ° C. to form fibrillated atelocollagen having a periodic fibrous structure as in a living body. Thereby, the physical properties are dramatically improved by a synergistic effect with the crosslinking treatment.

Further, by providing a silicone film layer 4 on the outermost layer, appropriate water vapor permeation is performed, so that the exudate is not stored on the wound surface and the wound surface is kept moist, while the protein component in the exudate leaks out. Is prevented, providing a highly favorable environment for tissue repair.

In addition, the antimicrobial agent 5 is contained in the silicone film, and the infection is prevented by continuously reducing the silicone film from the silicone film. Silver sulfadiazine (AgSD) was contained in the silicone film as an antibacterial agent at 10 mg, 20 mg and 30 mg, respectively, and the sustained release from the silicone film (20 μm thickness) was shown in FIG.

On the other hand, when the wound surface is already contaminated with bacteria, a large amount of the antibacterial agent is required, so that the antibacterial agent is desirably contained in either the collagen matrix layer 2 or 3.

 Hereinafter, the present embodiment will be specifically described.

Example 1 Preparation of Collagen Sponge Containing Antimicrobial Agent Phosphate buffer was added to a 0.3% atelocollagen solution at pH 3.0, and the mixture was placed in a thermostat at 37 ° C. for 4 hours to prepare fibrillated atelocollagen. While stirring 50 cc of fibrillated atelocollagen, 25 mg of silver sulfadiazine powder, 50 mg, 250 mg
And 500 mg were added and dispersed sufficiently, then poured into a styrene-type vat (10 cm × 10 cm) and freeze-dried. Further, the completed sponge was subjected to a crosslinking treatment under vacuum at 110 ° C. for 2 hours.

Example 2 Preparation of Antimicrobial-Containing Collagen Sponge Phosphate buffer was added to a 0.3% atelocollagen solution at pH 3.0, and the mixture was placed in a thermostat at 37 ° C for 4 hours to prepare fibrillated atelocollagen. This solution was freeze-dried into a sponge, and further immersed in a 0.01% hexamethylene diisocyanate ethanol solution for 1 day to crosslink. The crosslinked collagen sponge (10 cm × 10 cm) was immersed in 100 ml of an ammonia solution of silver nitrate (1 × 10 ⁻³ mol /).

Example 3 Preparation of Collagen Sponge Containing Antimicrobial Agent The collagen sponge obtained in Example 2 and immersed in the silver nitrate ammonia solution was further immersed in 100 ml of a sodium sulfadiazine solution (1 × 10 ⁻³ mol /).

Evaluation of antibacterial activity Muller-Hinton Agar (manufactured by Difco) was autoclaved, kept at 50 ° C, and dispensed in 20 ml aliquots into petri dishes.
It was left at room temperature for an hour to harden. After culturing the bacteria on a plate, various bacteria were suspended in a Tris buffer to form a bacterial solution, and the whole was applied to the medium three times with a cotton swab. Cut out the sample prepared by the above method to a diameter of 8 mm, place on a medium coated with bacteria,
The cells were cultured at 7 ° C for 18 hours. The results are shown in Table 1.

All the strains formed almost the same inhibition circle irrespective of the concentration at the combination of 0.25 mg / cm ² or more.

Example 4 Preparation of Artificial Skin Containing Antibacterial Agent In Example 1, 1.0 w of the fibrillated atelocollagen solution was used.
/ v%, a denatured atelocollagen solution was added, 25 mg of silver sulfadiazine was added with sufficient stirring, the mixture was poured into a sterol vat, and a linearized atelocollagen sponge cross-linked with 0.01% HDI was slowly added and freeze-dried. Next, a hexane solution of 50% Silastic silicone adhesive type A (Dow Corning) was applied on Teflon using a precision coating tool to form a film. Immediately after application, the sponge was placed and cured in an oven at 60 ° C. for at least 1 hour. Further evacuate under vacuum for 1 hour, raise the temperature to 110 ° C, keep vacuum for 2 hours, then lower the temperature to room temperature,
The sample was taken out to obtain an antibacterial agent-containing artificial skin.

〔The invention's effect〕

The present invention adheres to a wound surface because it has appropriate water vapor permeability and exudate absorption when applied to a wound, a skin wound, a skin abrasion wound, a traumatic skin defect wound, etc. In particular, by containing an antimicrobial agent, the antimicrobial agent is gradually released over a long period of time to prevent infection, and since the contact portion with the side has cell invasion, neutrophils and macrophages infiltrate early, Fibroblasts invade and are quickly replaced by their own tissue, promoting wound healing,
And reproduces neatly without leaving scars.

[Brief description of the drawings]

FIG. 1 is an enlarged sectional view showing a fine structure of an embodiment of the artificial skin of the present invention. DESCRIPTION OF SYMBOLS 1 ... artificial skin, 2 ... wound contact layer 3 ... support layer, 4 ... moisture permeation control layer 5 ... antibacterial agent FIG. 2: is a graph which shows the elution amount of silver sulfadiazine from a silicone membrane. The vertical axis is silicone film (5cm × 5c)
m, about 0.15 g) and the amount of silver sulfadiazine eluted in 100 ml of distilled water, indicating the sustained release of silver sulfadiazine.
The horizontal axis indicates time (day). -□-,-△-,-○-
Indicates the amount of each sulfadiazine silver contained in the silicone film.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Kaoru Koyamada 2656 Obuchi, Fuji-shi, Shizuoka Prefecture Terumo Corporation (72) Inventor Takeo Katakura 2656 Oobuchi, Fuji-shi, Shizuoka Prefecture Terumo Corporation

Claims (7)

(57) [Claims] 1. A wound contact layer comprising a matrix of fibrillar collagen and denatured collagen having a helix content of 0 to 80%, a support layer comprising a fibrillar collagen matrix having a cross-linked structure, and a moisture permeation control layer. An artificial skin which is sequentially laminated and at least one of the layers contains an antibacterial agent. 2. The artificial skin according to claim 1, wherein the antibacterial agent is silver sulfadiazine. 3. The artificial skin according to claim 1, wherein the antibacterial agent is gentamicin. 4. A fibrillar collagen and a helix content of 0.
8080% of a mixed solution with denatured collagen is placed in a container, a fibrous collagen matrix having a crosslinked structure is placed thereon, and lyophilized to form a porous body, and the upper surface of the fibrous collagen matrix is Place on a thin film of still permeable moisture permeable material spread on the substrate, dry until the thin film is cured, and finally apply
Heat treatment at 50 to 180 ° C. for 1 to 24 hours at less than Torr, containing an antibacterial agent in at least one layer of a mixed solution of fibrous collagen and denatured collagen, fibrous collagen matrix or moisture permeable substance A method for producing artificial skin, comprising: 5. The fibrillar collagen matrix is cross-linked by thermal dehydration or chemical treatment.
Method of producing artificial skin. 6. The thermal dehydration treatment is performed under a vacuum of 0.05 torr or less under a vacuum.
6. The method for producing artificial skin according to claim 5, wherein the artificial skin is heated in a temperature range of not less than 180C and not less than 180C. 7. The method according to claim 5, wherein the chemical treatment is a treatment with an aldehyde, carbodiimide, isocyanate or epoxy.