JPH0618583B2 - Artificial skin and its manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel artificial skin and a method for producing the same.

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

[Prior art and its problems] Temporarily applied to an affected area for the purpose of protecting the affected area due to a disease such as a burn, a skin-cutting wound and a traumatic skin defect wound, and pressure ulcer or a wound and promoting healing. Conventionally, gauze, absorbent cotton, etc. have been used as a wound dressing material, but this has a low ability to prevent bacterial infection, and since the exudate is absorbed promptly, the wound surface dries, causing pain and bleeding when removed. It was a companion. In addition, an ointment or the like is also used in combination, but in this case, on the contrary, the absorption of the exudate is insufficient and the wound surface becomes excessively wet.

Further, as an alternative to these, particularly when applied to a wide range of wound surfaces, silicone gauze, silicone rubber and nylon having a velor-like surface structure, artificial materials such as synthetic fiber sheets such as Teflon A coating film and a coating film of a bio-derived material such as freeze-dried pig skin, chitin nonwoven fabric, collagen film, polyamino acid sponge, mucopolysaccharide complex collagen film are also known. However, among these, the artificial coating film leaves various problems in terms of adhesion with the affected area, water vapor permeability, cracking, etc., while the coating film of a bio-derived material has characteristics such as biocompatibility. However, most of them have antigenicity, have defects such as bacterial infection and deterioration due to exudate, and have problems such as difficulty in obtaining raw materials. More recently, a composite membrane consisting of a collagen-treated nylon mesh and a silicone membrane has been developed and put into practical use, and it adheres well to the wound surface and has appropriate water permeability, but it adheres to the wound surface and the granulation tissue is covered. There was a drawback that it penetrated into the covering film.

[Problems to be Solved by the Invention] Conventional wound dressings have drawbacks as described above. Therefore, autologous transplantation is currently used as a treatment for an affected area when skin tissue is lost due to burns or the like. It is considered the best method. However, it is very difficult when the skin defect is large, and even if it is applicable, it is necessary to repeat transplantation repeatedly over a long period of time.

Therefore, it is desired to develop a wound dressing material that temporarily or permanently covers the affected area in place of autologous transplantation to prevent bacterial infection and body fluid outflow, and proliferates tissue cells to promote tissue repair. ing.

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

1) A wound contact layer composed of a matrix of fibrotic collagen and denatured collagen having a helix content of 0 to 80%, a support layer composed of a fibrotic collagen matrix having a crosslinked structure, and a water permeation control layer are sequentially laminated. Artificial skin that has been made.

2) The artificial skin according to the item 1, wherein the moisture permeation control layer is silicone.

3) The moisture permeation control layer has a thickness of 5 to 200 μm 1
Item or artificial skin according to item 2.

4) A mixed solution of fibrillated collagen and denatured collagen having a helix content of 0 to 80% is placed in a container, a fibrillated collagen matrix having a crosslinked structure is placed thereon, and freeze-dried to form a porous body. , Placing the upper surface of the fibrillated collagen matrix on a thin film of a moisture permeable material that is still tacky and has been spread over a substrate, dried until the thin film hardens, and finally under a vacuum of less than 0.05 Torr A method for producing artificial skin, which comprises heat treating at 50 to 180 ° C. for 1 to 24 hours.

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

6) Thermal dehydration treatment under vacuum of 0.05 Torr or less, 50 ℃ or more 18
The method for producing artificial skin according to item 5, which comprises heating in a temperature range of 0 ° C. for 24 hours or more.

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

As described above, the artificial skin of the present invention comprises the wound contact layer (lower layer), the support layer (middle layer) and the water permeation control layer (upper layer).

The wound contact layer covers the wound surface directly and protects it softly, sores, moisturizes and prevents bacterial contamination. Furthermore, when applied to the wound surface, the wound contact layer infiltrates macrophages, neutrophils, and other inflammatory cells very early, and then fibroblasts and the capillary system infiltrate prematurely, resulting in dermis. 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 water permeation regulating layer thereon are demarcated and excluded. As a material constituting the wound contact diagram, fibrotic collagen, denatured collagen having a helix content of 0 to 80%, and a matrix are preferable.

Since the artificial material using collagen is a biological material, it is considered that it has a high affinity for cells and tissues, but it is easily decomposed and absorbed in the living body. In using it, we introduce crosslinks by some means,
It is necessary to strengthen the physical properties. As the cross-linking method, dehydration cross-linking by heating, chemical cross-linking using a chemical or the like can be adopted. Of these, thermal dehydration cross-linking is safer than chemical treatment, but physical resistance to collagenase / enzyme is physically lower than chemical cross-linking. Therefore, a method of using chemical crosslinking in combination with thermal crosslinking or using chemical crosslinking alone is selected. When this is carried out, the physical properties are remarkably improved. For example, 11
Even if a thermal cross-link is introduced by placing it under vacuum at a temperature of 0 ° C for 24 hours, it will dissolve within 1 day if it is allowed to stand at 37 ° C in collagenase 3 unit / m, but only isocyanate-based cross-linking will occur. The applied sample is 3 in 100 unit / m of collagenase.
No change in morphology is observed even if left at 7 ° C for 7 days. However, when a strong crosslink is introduced when it is used as artificial skin, the affinity of collagen before the introduction for cells and tissues is significantly reduced, and macrophages, neutrophils and other inflammatory substances are present in the collagen matrix. Cells and fibroblasts are almost absent, and eventually the matrix is eliminated to form epidermis, so-called down growth, and inflammatory granulation is formed. On the other hand, collagen with good cell invasion
In the denatured collagen matrix, neutrophils and macrophages infiltrate early and fibroblasts invade, but wound contraction occurs like open wounds. In other words, it is a contradictory phenomenon that it is difficult to simultaneously achieve both enhancement of physical properties and improvement of biological performance such as affinity for cells and tissues, and a satisfactory wound dressing or artificial skin could not be conventionally obtained.

As a result of intensive studies, the present inventors have found that the combination of fibrotic collagen with denatured collagen having a helix content of 0 to 80% achieves both enhancement of the physical properties of collagen and improvement of biological performance. I found a thing.

Crosslinking of collagen is carried out by heating collagen or treating it with a crosslinking agent according to a conventional method.

In the case of heat treatment, collagen is dehydrated by holding under vacuum at less than 0.05 Torr at 50 to 180 ° C. for 1 to 24 hours. It is preferably heated under vacuum at less than 0.05 Torr at 100-120 ° C. for 2-8 hours.

When treated with a crosslinking agent, the crosslinking agent is not particularly limited,
Aldehyde crosslinking agents such as glutaraldehyde, isocyanate crosslinking agents such as hexamethylene diisocyanate, carbodide crosslinking agents such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, and the like are used.

If the degree of cross-linking is too low, sufficient physical strength as a medical material cannot be obtained, and if it is too high, the structure and properties of collagen are impaired, so this should be avoided. 0.01-5% (w / v),
Preferably, crosslinking is carried out at a concentration of 1 to 3% (w / v) of a crosslinking agent to obtain collagen having an appropriate degree of crosslinking.

As for the collagen to be crosslinked, the dispersed water-soluble collagen having triple-stranded helix does not improve the physical properties so much even if it is crosslinked. Therefore, disperse the dispersed collagen at 37 ° C using a phosphate buffer. It is preferably subjected to a summing treatment to form a reconstituted fibrillated collagen having a periodic fibrous structure as in vivo. As a result, the physical properties are dramatically improved by the synergistic effect with the crosslinking treatment.

On the other hand, for denatured collagen having a helix content of 0 to 80%, collagen derived from bovine dermis is treated with an acid or an alkali, and the obtained collagen aqueous solution having a triple chain helix is heated at 37 to 90 ° C in the presence of water. Obtained by The above-mentioned raw material collagen for fibrosis and denaturation is preferably one in which acid or alkali-treated collagen is further digested to remove telopeptides less than its molecule with proctase or pepsin to eliminate antigenicity.

The degree of denaturation of collagen is indicated by the content of helix structure. The helix content is a triple-stranded helix content peculiar to collagen. In denatured collagen, this helix is randomly coiled, so the helix content corresponds to the degree of denaturation. This helix content can be measured by circular dichroism or an infrared spectrophotometer (PLGord
on, IVYannas, et al, Macromolecules, 7 (6) 954 (1974),
Nakakura, Hashimoto et al., Polymers 41 (8) 473 (1984)). The index of the denaturation degree of collagen used here, that is, the helix content is 0 to 80%, and more preferably 0 to 50%. For example, when the collagen solution is heat-treated at 60 ° C for 30 minutes, the helix content is about 40%, and when it is heat-treated at 100 ° C for 24 hours, the helix content becomes 0, and it can be seen that a part of the collagen molecule is cleaved by electrophoresis. . The composition of denatured collagen in the matrix is 5-80%
And more preferably 10 to 50%.

The wound contact layer is obtained by mixing the aqueous solution of fibrotic collagen with the aqueous solution of denatured collagen and freeze-drying.

In the artificial skin of the present invention, the support layer reinforces the mechanical strength of the wound contact layer and smoothes the cell invasion property.

As described above, the wound contact layer has resistance to collagenase, but the mechanical strength as a covering material is not sufficient, and eventually it becomes assimilated with the living body,
A support layer is needed to protect this from external stimuli. Therefore, the material of the support layer is required to have a certain level or more of mechanical strength, but at the same time, it must not inhibit cell invasion into the wound contact layer. Fibrillated collagen having a crosslinked structure is mentioned as one that satisfies these conditions.

As the fibrous collagen having a crosslinked structure used as the support layer, the same crosslinked fibrotic collagen used in the wound contact layer can be used.

The water permeation control layer is a layer for controlling water in a wound when the artificial skin is applied to the wound surface.

The moisture permeation control layer performs appropriate water vapor permeation, exudate is not stored on the wound surface, and the wound surface is kept in a wet state, on the other hand, leakage of protein components in the exudate to the outside is prevented, It provides a very favorable environment for tissue repair.

It is conventionally known to provide a water permeation control layer on artificial skin, and known materials can be used in the present invention. That is, a layer of non-toxic material having a water flux of about 0.1 to about 1 mg / cm ² / hour is used. Thickness is 5
200 μm is suitable. As the non-toxic material, silicone resin, polyacrylate ester, polymethacrylate ester and polyurethane are used, and silicone is particularly preferable.

The artificial skin of the present invention is prepared by placing a mixed solution of fibrotic collagen and denatured collagen having a helix content of 0 to 80% in a container, placing a fibrotic collagen matrix having a crosslinked structure on it, and lyophilizing the mixture. Form a porous body,
The upper surface of the fibrillated collagen matrix is placed on a thin film of water-permeable material still sticky that has been spread on a substrate, dried until the thin film hardens, and finally under vacuum at less than 0.05 Torr. It is produced by heat treatment at 50 to 180 ° C. for 1 to 24 hours.

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

1) After preparing an aqueous collagen solution, a porous sponge is prepared by a freeze-drying method. Then, a chemical crosslinking reaction is performed.

2) Prepare an aqueous collagen solution. This is divided into two, one is left as it is, and the other is denatured collagen by denaturing this collagen aqueous solution by heat treatment or the like. After mixing both solutions, they are poured into a predetermined container, and the crosslinked collagen sponge prepared in 1) above is slowly placed on the upper layer to prepare a porous sponge having a two-layer structure by a freeze-drying method.

3) Next, a solution of a water-permeable substance such as silicone is spread on Teflon to form a film, and in a state where the film still has tackiness, the chemically crosslinked collagen matrix layer obtained in the above step is Place it so that the upper surface adheres to the film, dry it, and further subject it to heat treatment,
The membrane is cured and a moisture permeation control membrane is attached to the upper surface of the support layer.

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

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

Example 1) Preparation of Fibrotic Atelocollagen 1.0 g of atelocollagen was dissolved in dilute hydrochloric acid of pH 3.0 to 0.3 w /
v%. This solution was placed in a constant temperature bath at 4 ° C. with stirring to add a phosphate buffer solution to prepare a collagen solution having a final concentration of 0.1 w / v% atelocollagen, 30 mM 2-sodium phosphate and 100 mM sodium chloride. Then 37
Immerse in a constant temperature bath at ℃ for 1 day, and fibrillated atelocollagen (F
C) A solution was prepared. This solution was rapidly frozen at -30 ° C and then freeze-dried to prepare a sponge.

2) Preparation of denatured atelocollagen solution 1.0 g of atelocollagen was dissolved in dilute hydrochloric acid of pH 3.0.
After treating this solution in a constant temperature bath at 60 ° C for 30 minutes,
After allowing to stand for a time, a denatured atelocollagen (HAC) solution was obtained. The helix content of the denatured atelocollagen thus obtained was about 40%.

3) Preparation of fibrillated atelocollagen-denatured atelocollagen matrix 0.3 w / v% fibrillated atelocollagen (F
C) and 1w / v% denatured atelocollagen (HAC) at 37 ℃
And mixed for 1 hour. This solution was rapidly frozen at -30 ° C and then freeze-dried to prepare a sponge.

4) Thermal dehydration cross-linking of fibrotic atelocollagen matrix The product of 1) above is evacuated under a vacuum of less than 0.05 torr for 1 hour, further lowered to 110 ° C and kept in vacuum for 2 hours, and then lowered to room temperature. , The sample was taken out.

5) Isocyanate Crosslinking of Fibrotic Atelocollagen Matrix The product of 1) above was crosslinked by immersing it in an ethanol solution of 0.01% hexamethylene diisocyanate (HDI) at room temperature for 24 hours. Further, in order to remove uncrosslinked isocyanate, washing with water was carried out several times and freeze-drying was performed.

6) Thermal dehydration cross-linking of fibrotic atelocollagen-denatured atelocollagen matrix The product of 3) above is evacuated for 1 hour under a vacuum of less than 0.05 Torr, further lowered to 110 ° C. for 2 hours or 24 hours.
The vacuum was maintained for a period of time, then the temperature was lowered to room temperature, and the sample was taken out.

Test Example 1 In Vivo Subcutaneous Implantation Test of Various Collagen Matrices The matrix obtained above was subcutaneously implanted into rats,
The histology was searched pathologically. Approximately 200 g for subcutaneous implantation
Wistar-KY strain, female rats were used. Before embedding, after anesthetizing with 5-fold diluted Nembutal, the back of the rat was wet with isodine solution for surgery (manufactured by Meiji Seika Co., Ltd.), and the back was carefully shaved so as not to leave uncut hair with a razor for shaving. . Then, the shaved back surface was disinfected with isodine and ethanol. From each incision, the incision was widened to create a void within the loose connective tissue beneath the cutaneous muscle of the rat. The sample was put into this void so that the entire sample lay flat.
The cut end was sutured with a nylon thread with a square needle. Three stitches were cut at the cut end. The same specimen was similarly implanted in another rat.
At 3 and 28 days after implantation, the animals were killed with ether or 2-fold diluted Nembutal. Make sure that the embedded sample remains in the tissue, and cut the skin tissue on the rat back muscle 8 cm ×
Cut to 12 cm or larger. This organization
The cells were placed in a 10% neutral buffered formalin solution, left to stand overnight for fixation, and then subjected to histopathological search.

The tissue was cut into pieces of about 0.5 cm x 2.5 cm to ensure that the sample was contained. This was clarified with ethanol and then with xylene, and finally replaced with paraffin. After replacement,
Place the tissue containing the sample in the heated solution of solid paraffin,
It was rapidly cooled to complete the paraffin embedding. The embedded tissue is sliced with a rotary microtome manufactured by Yamato Corporation,
Paraffin sections with a thickness of 4 μm were prepared. After deparaffinizing this, pathological tissue staining was performed by an arbitrary staining method to prepare a preparation. As the pathological tissue stain, hematoxylin-eosin (HE) stain, Azan stain, resorcin-fuchsin stain and the like were adopted. The results are shown in Table 1.

With FC alone, neutrophil infiltration was strong on day 3 and fibroblast invasion was moderate, and on day 28,
The resulting granulation tissue was atrophied. On the other hand, H
By containing 10 to 20% by weight of AC, the neutrophil infiltration on the 3rd day was weak, and conversely, the invasion of fibroblasts was further improved. Furthermore, it was revealed that the atrophy of the granulation tissue on the 28th day was remarkably alleviated.

7) Preparation of artificial skin In the above 3), the fibrotic atelocollagen concentration was set to 1.0 w / v.
%, And the mixed solution of fibrotic atelocollagen-denatured atelocollagen is injected into a stainless steel vat, and the sponge of the fibrotic atelocollagen matrix cross-linked with 0.01% HDI prepared in 5) above is slowly put into solution. Float on the upper layer. -30 in this state
After rapid freezing to ℃, freeze well and freeze-dry under vacuum below -40 ℃ / 0.1 Torr, a sponge with a bilayer structure consisting of fibrillated atelocollagen matrix and fibrillated atelocollagen-denatured atelocollagen matrix was obtained. . Next, a 50% Silastic silicone adhesive type A (Dow Corning) hexane solution was applied onto Teflon using a precision coating tool (applicator) to form a film. Immediately after application, place the above sponge so that the fibrotic atelocollagen matrix is on the silicone side, and at room temperature.
After leaving for about 10 minutes, it was cured in an oven at 60 ° C. for at least 1 hour. A vacuum of less than 0.05 Torr was further applied for 1 hour, the temperature was further raised to 110 ° C., the vacuum was maintained for 2 hours, then the temperature was lowered to room temperature, and a sample was taken out to obtain artificial skin.

Test Example 2 Transplantation test of artificial skin to rat skin defect wound The matrix obtained above was transplanted to the back skin of a rat and tested. Wistar-KY rats (200-400 g) were shaved under Nembutal anesthesia, and a 20 × 20 mm total wound skin defect with a cutaneous muscle as the wound surface was prepared on the rat dorsal skin disinfected with isodine, and after hemostasis and drying, The samples containing raw food were attached to each. The edges of the silicone membrane were ligated and fixed with 16 sutures. On top of that, 4 sheets of Solfrene (made by Terumo) were stacked, and then wrapped with a stretchable bandage such as elasticon to be wound and fixed with pressure. The observation was carried out 1, 2, 3 and 4 weeks later, and autopsy was carried out 4 weeks after the transplantation, and the results of histopathological search were shown in Table 2.

EFFECT OF THE INVENTION According to the present invention, the content of fibrotic collagen and helix is 0.
There is provided artificial skin in which a wound contact layer composed of a matrix with denatured collagen of -80%, a support layer composed of a fibrotic collagen matrix having a crosslinked structure, and a water permeation control layer are sequentially laminated.

The artificial skin of the present invention is applied to a damaged surface when the skin is damaged by a wound, a burn, a pressure sore, etc., and softens and protects the wound surface, relieves pain, and prevents bacterial infection.

Furthermore, since the wound contact surface of the artificial skin of the present invention has cell invasion properties, when applied to the wound surface, fibroblasts invade the wound contact layer at an early stage to build a dermis-like connective tissue. Healing is promoted. When the artificial skin of the present invention is applied, the wound surface is healed cleanly without leaving scars.

Further, according to the present invention, an advantageous method for producing the artificial skin is provided.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kaoru Oyamada One of the 2656 Obuchi, Fuji City, Shizuoka Prefecture Terumo Co. Ltd.

Claims (7)

[Claims] 1. A wound contact layer comprising a matrix of fibrillated collagen and denatured collagen having a helix content of 0 to 80%, a support layer comprising a fibrillated collagen matrix having a crosslinked structure, and a water permeation control layer. Artificial skin that is sequentially laminated. 2. The artificial skin according to claim 1, wherein the moisture permeation control layer is made of silicone. 3. The artificial skin according to claim 1, wherein the water permeation control layer has a film thickness of 5 to 200 μm. 4. Fibrotic collagen and helix content of 0
~ 80% mixed solution with denatured collagen is placed in a container, a fibrotic collagen matrix having a crosslinked structure is placed thereon, and freeze-dried to form a porous body, and the upper surface of the fibrotic collagen matrix is Place on a thin film of moisture permeable material still sticky, spread on a substrate, dry until the thin film hardens, and finally under vacuum 0.05
A method for producing artificial skin, which comprises heat-treating at 50 to 180 ° C. for less than 1 to 24 hours. 5. The fibrillated collagen matrix is crosslinked by heat dehydration or chemical treatment.
Method for producing artificial skin. 6. The thermal dehydration treatment is performed under a vacuum of 0.05 Torr or less.
The method for producing artificial skin according to claim 5, wherein the heating is performed in a temperature range of 1800C to 180C for 1 to 24 hours. 7. The method for producing artificial skin according to claim 5, wherein the chemical treatment is a treatment with aldehyde, carbodiimide, isocyanate or epoxy.