JPH04135564A - Artificial skin and production thereof - Google Patents

Abstract

PURPOSE:To softly protect a wound surface and to prevent the infection with bacteria by forming the part in contact with the wound part of a collagen/ denatured collagen matrix having good cell infiltration property and a steam permeation control layer in tight contact with a wound contact layer. CONSTITUTION:The artificial skin 1 consists of the collagen/denatured collagen matrix layer 2 having the good cell infiltration property and the steam permeation control layer 3 adjacent to the wound contact layer 2 in the part in contact with the wound part. The wound contact layer 2 which has the cell infiltration property refers to such layer that, when the artificial skin is applied to the wound part, inflammatory cells, such as microphages and neutrophil, infiltrate into the artificial skin and fibroblasts infiltrate into the skin in an early period and eventually, the true skin-like bond and tissue are rebuilt. The denatured collagen used in the wound contact layer 2 is formed by subjecting the collagen to a heating treatment, chemical treatment, physical treatment, etc. The heat treatment is most preferable. The wound surface is softly protected in this way and the pains are relieved. In addition, the infection with the bacteria is prevented.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applied to the damaged skin area when the skin is damaged due to a wound, burn, etc., and is used to soften and protect the damaged skin area without drying it. and relates to artificial skin that replaces the patient's own tissue at an early stage.

[Conventional technology]

Traditionally, gauze has been used as a wound dressing material temporarily applied to the affected area in order to protect the affected area due to diseases or wounds such as burns, skin harvest wounds, skin abrasion side, traumatic skin defect side, etc. and promote healing. , absorbent cotton, etc., was used, but this had low ability to prevent bacterial infection, and because it quickly absorbed exudate, the wound surface dried, causing pain and bleeding when removed. . In addition, ointments and the like have also been used, but in this case, the exudate is not sufficiently absorbed and the wound surface becomes excessively moist.

Alternatively, artificial materials such as silicone gauze, silicone rubber, and synthetic fiber sheets such as nylon and Teflon with a velour-like surface structure can be used as alternatives to these, especially when the wound surface is extensive. Coating membranes and coating membranes made of biologically derived materials such as freeze-dried pork skin, fibrin membranes, collagen membranes, polyamino acid sponges, and mucopolysaccharide complex collagen membranes are also known. However, among these, artificial coating membranes have poor adhesion to the affected area,
Various problems remain in terms of water vapor permeability, cracking, etc. On the other hand, coating membranes made of biological materials have characteristics such as biocompatibility, but many of them are antigenic and are susceptible to bacterial infection. , it has disadvantages such as deterioration due to exudate, and there are also problems such as difficulty in obtaining raw materials. Furthermore, recently,
A composite membrane consisting of a collagen-treated nylon mesh and a silicone membrane has been developed and put into practical use, and although it adheres well to the wound surface and has moderate water permeability, it adheres to the wound surface and granulation tissue remains in the covering membrane. It had the disadvantage of getting into the

From this point of view, wound dressings or artificial skin generally have: ■) Adhesiveness to the wound area to prevent infection and pain, and 2) Appropriate water vapor permeability to provide appropriate moisture to the wound surface. 3) Regenerates good granulation tissue, 4) Prevents leakage of protein components in body fluids, 5) Raw materials are easily available and manufacturing is simple. Although it is desired that these conditions be met, the current situation is that wound dressings and artificial skin that fully satisfy these conditions have not yet been obtained.

[Problem that the invention seeks to solve]

Artificial materials using collagen are biologically derived materials, so although they are thought to have a high affinity for cells and tissues, they are easily decomposed and absorbed within living organisms. In order to use it, it is necessary to introduce crosslinking by some means to strengthen its physical properties. As the crosslinking method, dehydration crosslinking by heating, chemical crosslinking using chemicals, etc. can be employed. Among these, thermal dehydration crosslinking is safer than chemical treatment, but it is physically less resistant to collagenase and enzymes than chemical crosslinking. Therefore, methods are selected in which chemical crosslinking is used in combination with thermal crosslinking, or chemical crosslinking is used alone. When this is carried out, the physical properties are significantly improved.

For example, when thermal cross-linking is introduced by placing it under a steam atmosphere at a temperature of 110°C for 24 hours, collagenase 3 units/ml
When left standing at 37°C in a liquid solution, it dissolves within one day, whereas in a sample subjected to only isocyanate crosslinking, no change in morphology was observed even after 7 days at 37°C in 100 units/ml of collagenase. . However, when strong crosslinks are introduced when used as artificial skin, the affinity of collagen for cells and tissues that it had before introduction is significantly reduced, and macrophages, macrophages, etc.
Inflammatory cells such as neutrophils invade and inflammatory granulation is formed, or the matrix is eliminated by so-called downgrowth of the regenerated epidermis without even inflammatory cells invading. On the other hand, collagen with good cell penetration
In the denatured collagen matrix, neutrophils and macrophages infiltrate early, followed by fibroblasts, but wound contraction occurs as in the open side. In other words, strengthening physical properties and improving biological performance, such as affinity for cells and tissues, are contradictory phenomena that are difficult to reconcile, and it has not been possible to find a satisfactory wound dressing or artificial skin.

In view of the above circumstances, the present inventors have already developed an artificial skin with a three-layer structure consisting of a collagen matrix layer with good cell penetration, a sufficiently cross-linked collagen support layer, and a water vapor permeation regulating layer. (Unexamined Japanese Patent Publication No. 2-34185). In animal experiments, it was observed that when this artificial skin was applied to a wound surface, fibroblasts quickly invaded the wound contact layer and built a dermis-like connective tissue, promoting wound healing. However, when this artificial skin is applied to extensive burn wounds or extensive skin defects, it takes a very long time to heal because there is a limit to the extension of the epidermis. In this case, it is possible to use split-thickness skin grafting, which is generally practiced clinically, and after transplanting the above artificial skin for a certain period of time, a supporting layer that has been sufficiently cross-linked with the water vapor permeation regulating layer is used. 2
By peeling off the layer and performing a split-thickness skin graft, survival can be achieved.
It has become possible to apply it to a wide range of defective areas. However, although it is rare, the supporting layer having a crosslinked structure may not be completely peeled off and may remain on the collagen-denatured collagen matrix layer, affecting the engraftment of the split-thickness skin graft.

[Means to solve the problem]

The present invention is an artificial skin characterized in that a region that can come into contact with a wound is composed of a collagen-denatured collagen matrix with good cell penetration, and a water vapor permeation regulating layer that is in close contact with the wound contact layer.

The present invention also provides an artificial skin in which the wound contact layer is made of a denatured collagen matrix with good cell penetration, and the denatured collagen is bonded to the collagen skeleton by thermal dehydration crosslinking. The denatured collagen has a triple-chain helix content molecularly unique to collagen of 0 to 80%, preferably 0 to 50%, more preferably 30 to 50%, and the denaturation is performed by heat treatment, chemical treatment, physical treatment, etc. However, heat denaturation treatment is most preferred.

The density of collagen-denatured collagen is not particularly limited, but is preferably in the range of 0.01 to 0.1 g/rIj, more preferably in the range of 0.03 to 0.06 g/cffl. Under these conditions, even if the intermediate layer described in JP-A-2-34165 is not present, it can withstand strength.

Thermal dehydration crosslinking is performed under vacuum at temperatures below 0.05 Torr.
The temperature is 0 to 180° C. for 1 hour or more and 24 hours or less, but preferably the temperature is 100 to 120° C. under vacuum and less than 0.05 torr for 2 hours or more and 8 hours or less.

The present invention also provides an artificial skin in which the water vapor permeation regulating layer is a silicone elastomer thin film or a polyurethane elastomer thin film. The present invention further provides an artificial skin in which the water vapor permeation regulating layer is a thin film of 5 to 2001 t1m.

Water vapor transmission rate is 10-5000 g/rd・24
hr (JIS standard) is preferable.

[For production]

The present invention will be explained in detail based on embodiments. FIG. 1 is an enlarged sectional view showing the fine structure of one embodiment of the artificial skin of the present invention. As shown in FIG. 1, the artificial skin 1 of the present invention includes a collagen-denatured collagen matrix layer 2 with good cell penetration, and a wound contact layer 2 that can come into contact with a wound.
It consists of a water vapor permeation regulating layer 3 adjacent to the water vapor permeation regulating layer 3.

Here, what means that the wound contact layer 2 has cell-invasive properties is as follows:
When the artificial skin 1 is applied to a wound, inflammatory cells such as macrophages and neutrophils infiltrate, and fibroblasts invade at an early stage, resulting in the reconstruction of dermal-like connective tissue. That's true. The denatured collagen used in the wound contact layer 2 may be subjected to heat treatment, chemical treatment, physical treatment, etc., but heat treatment is most preferred. The degree of denaturation of collagen is indicated by the content of helical structures. The helix content refers to the triple-chain helix content unique to collagen, and since the helices in denatured collagen are randomly coiled, the helix content corresponds to the degree of denaturation. This helical content can be measured using circular dichroism or infrared spectrophotometry (P, L, Gordon
, 1. V, Yannas, etal, Mae
romolecules, 7 (6) 954 (1
974), Nakura.

Hashimoto et al., Polymer Papers 41 (8) 473 (198
4)). An indicator of the degree of collagen denaturation used here,
Already, the helix content is 0-80%, preferably 0-50%, more preferably 30-40%. For example, when a collagen solution is heat-treated at 60°C for 30 minutes, the helix content is approximately 40%, and when heat-treated at 100"C for 124 hours, the helix content becomes O, and electrophoresis shows that some of the collagen molecules are cleaved.

The composition of denatured collagen is 5 to 80%, more preferably 10 to 50%. The collagen to be used is preferably atelocollagen from which antigenic determinants have been removed by enzymes in order to suppress antigenic expression.If atelocollagen is used as it is in a dispersed state, the physical properties will not improve much even if cross-linking is introduced, so atelocollagen is 37 It is more preferable to perform a neutralization treatment (using a phosphate buffer) at a temperature of 0.degree. C. to form fibrotic atelocollagen having a peripheral fibrous structure similar to that found in vivo.

Furthermore, by providing the water vapor permeation control layer 3, appropriate water vapor permeation is achieved, so that exudate does not accumulate on the wound surface and the wound surface is kept moist, thereby preventing protein components in the wound exudate from leaking to the outside. is prevented, providing a highly favorable environment for tissue repair.

The artificial skin of the present invention can be easily manufactured, for example, as follows.

Prepare a collagen aqueous solution. This is divided into two parts, one part is left as is, and the other part is made into denatured collagen by denaturing this aqueous collagen solution by heat treatment or the like. After both solutions are mixed, they are poured into a predetermined container, and a collagen sponge made of a collagen-denatured collagen matrix is produced by freeze-drying.

Next, a solution of water vapor permeable material such as silicone is spread on Teflon to form a film, and while the protective film is still sticky, the collagen matrix obtained in the above step is placed on it, and after drying at room temperature. Further, if necessary, heat treatment is performed to harden the protective film.

Finally, thermal dehydration crosslinking is performed under vacuum, and the composite layer is further peeled off from the substrate to obtain the desired artificial skin.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Preparation of fibrotic atelocollagen denatured-atelocollagen mixed solution Atelocollagen (manufactured by Kouken Co., Ltd.) was dissolved in dilute hydrochloric acid of I) H3,O at a temperature of 4°C to 0.3 to 0.4 w/v.
%. This solution was sterilized by filtration by sequentially passing it through two types of filters with pores of 0.8 and 0.2 diameter, and then a phosphate buffer solution with a pH of 7.4 was added to the solution while stirring while maintaining the solution at 4°C. In addition, the final concentration is 0.1 to 0.15 ν/V%
Atelocollagen (30mM sodium phosphate, 110
0ff1 sodium chloride) was used as a collagen solution. The mixture was then left in a constant temperature bath at 37° C. for 4 hours to prepare a fibrotic atelocollagen (FC) solution. And this FC
Concentrate the solution by centrifugation under sterile conditions to a concentration of 4.
Adjusted to w/v%.

On the other hand, 0, 3 to 0.
A 4 w/v% atelocollagen solution was freeze-dried, redissolved in sterile distilled water to a concentration of 6.8 v/v%, and left in a constant temperature bath at 60°C for 30 minutes to cause thermal denaturation. A denatured atelocollagen (HAC) solution was prepared. This HAC solution was sterilized by filtration at a temperature of 37°C through a filter with pores of 0.45 us diameter, and then added to the above-mentioned 4 v/v% FC solution with HAC/FC+HAC-0.
, 1. HAC for 12 volumes of Fe5O
(approximately 67 volumes) and stirred to obtain a mixed solution of fibrotic atelocollagen-denatured atelocollagen (FC-HAC).

<Preparation of artificial skin> The mixed solution of FC-HAC of Example 1 was injected into a stainless steel pad, rapidly cooled to below -30°C and sufficiently frozen, and then vacuumed at -40°C below 10.1) -l. A matrix of FC-HAC was obtained by freeze-drying under. Next, a hexane solution of 67% medical grade Silastics silicone (adhesive silicone type A, manufactured by Dow Corning Corporation) was applied onto the Teflon plate using a precision coating tool (applicator) to form a film.
The FC-HAC matrix described above was placed on top of the wet layer immediately after coating, left at room temperature for 10 minutes, and then cured in an oven at 60° C. for at least 1 hour. Another 0.0
5) The artificial skin of the present invention was created by applying a vacuum for 1 hour under a vacuum of less than 100 ml, raising the temperature to 110°C and keeping the vacuum for 2 hours, and then lowering the temperature to room temperature.

<Animal experiment> Transplantation test of artificial skin to rat skin defect side The matrix obtained above was transplanted to the back skin of a rat and tested. W 1star -K Y rat (200-400
g) A 20 x 20 mm wound was made on the subcutaneous fascia of the rat's dorsal skin, which was dehaired under Nembutal anesthesia and disinfected with isodine.
A complete skin defect wound was created, and after the bleeding was stopped and dried, a sample containing saline was applied to each wound. The edges of the silicone membrane were ligated and fixed at 16 locations with sutures. On top of that, four sheets of Solfrane (manufactured by Terumo) were layered, and an elastic bandage such as Elasticon was further wrapped around the body and fixed under pressure. At 1, 2, and 3 weeks after transplantation, the upper silicone layer was peeled off, and an autologous split-thickness skin graft was transplanted onto the FC-HAC layer and observed for an additional 4 weeks. Macroscopically, not much shrinkage was observed, and the split-thickness skin graft had not detached. Histopathologically, the boundary between the dermis-like tissue regenerated using the artificial skin and the engrafted split-thickness skin graft was clearly distinguishable, but an integrated, good granulation tissue was observed.

〔Effect of the invention〕

According to the present invention, the content of fibrotic collagen and hesox is 0.
An artificial skin is provided in which a wound contact layer consisting of a matrix with ~80% denatured collagen and a water vapor permeation regulating layer such as a silicone elastomer having water vapor permeability are laminated in this order.

The artificial skin of the present invention is applied to the injured surface when the skin is damaged due to wounds, burns, bedsores, etc., to soften and protect the wound surface, relieve pain, and prevent bacterial infection.

Furthermore, the wound contact surface of the artificial skin of the present invention has cell-invading properties, so when applied to the wound surface, fibroblasts quickly invade the wound contact layer and build a dermis-like connective tissue, so that the wound contact surface is resistant to cell invasion. Pain relief is promoted.

Furthermore, after being transplanted and maintained for a certain period of time, the upper water vapor permeation regulating layer is peeled off and an autologous split-thickness skin graft is transplanted, and the graft can also survive.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view showing the fine structure of an embodiment of the artificial skin of the present invention. 1...Artificial skin 2...Cell-penetrating support layer 3...Water vapor permeation regulating layer

Claims (2)

[Claims] (1) An artificial skin comprising a matrix support layer made of collagen and denatured collagen containing at least 5% by weight of denatured collagen, and a water vapor permeation regulating layer laminated thereon. (2) A mixed solution of collagen and denatured collagen containing at least 5% by weight of denatured collagen is poured into a container and freeze-dried to form a matrix, which is further spread on a substrate that is still sticky and water vapor permeable. The matrix is placed on a thin film of material, dried until the film is cured, and finally heated under a vacuum of less than 0.05 Torr for 50 to
A method for producing artificial skin, the method comprising heating at 180°C for 1 to 24 hours.