JP2000262610A - Artificial corium for private extraction hair foliculus transplantation designed for epidermis regeneration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an artificial corium useful for the treatment of a wide rang of full-thickness skin deficiency wounds as the artificial corium enables the complete recovery thereof in a short period of time without the execution of flap skin collection or skin collection which is the heavy burden to a patient and without the need for a high degree of culture technique and equipment. SOLUTION: This artificial corium for private extraction hair foliculus transplantation consists of a wound contact layer consisting of a composite matrix of fibrillated atherocollagen derived from the mammals and the denatured atherocollagen of 0 to 80% in helix content and a moisture permeation control layer, and contains slits of 3 to 10 mm in length and 10 to 5,000 μm in width penetrating at the wound contact layer at 10 to 10,000 pieces per 100 cm2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an artificial dermis useful for treating a wide range of full-thickness skin defect wounds such as burns, pressure ulcers, skin ulcers, surgical wounds and trauma.

[0002]

2. Description of the Related Art Conventionally, burns, pressure ulcers, skin ulcers, surgical wounds,
In the treatment of a wide range of full-thickness skin defect wounds such as trauma, the skin was reconstructed by flaps or skin grafts collected from the patient. In addition, skin reconstruction using a combination of artificial dermis and stratified skin grafting has also been performed. However, in both cases, the burden on the patient of flap sampling or skin removal (specifically, the occurrence of new percutaneous wounds and surgery with anesthesia)
There was a drawback of forcing. In addition, since the area where the skin can be cut is limited, in cases where the skin defect is extensive, such as a whole-body burn, the thin stratified skin graft must be harvested from the same skin area many times. It takes two weeks or more for the skin to be harvested to heal and allow skin to be harvested again, so that it takes a very long time to complete the treatment.

On the other hand, attempts have been made to culture epidermal cells derived from a patient by using a cell culture technique which has been highly developed in recent years, and to transplant a “cultured epidermal sheet” in which the epidermal cells are layered into a full-thickness skin defect wound. Has been done. However, the epidermis interacts with mesenchymal tissues such as the dermis, and it has been theorized that a cultured epidermis sheet does not "engraft" in a full-thickness skin defect wound where no dermis component exists. I have. Therefore,
The idea of transplanting the “cultured epidermal sheet” onto the artificial dermis, a matrix such as collagen and cells such as fibroblasts and epidermal cells are co-cult in vitro.
ure (complex culture), and an idea of seeking the source of cells at that time from the outer root sheath cells derived from the extracted hair follicle (Ann. Plas).
t. Surg. , 33 [3] (1994) Kurat
a, S. et al. , P. 290-294) have been proposed, but all have the disadvantage that they require advanced culturing techniques and equipment and time for culturing.
Japanese Patent No. 2722838 discloses that a method for producing a specific skin substitute includes "transplanting at least one hair follicle or a fragment of a hair follicle extracted from human or animal skin into a dermis substitute substrate" or the like. A method for producing a skin substitute characterized by the following has been proposed. In the manufacturing method,
In vitro, a hair follicle containing keratin-synthesizing cells is inserted into a substrate, the keratin-synthesizing cells are cultured for a certain period of time while immersed in a nutrient medium, and then cultured in contact with air. Once removed, keratinocytes are grown and a skin substitute is obtained. However, the method does not
The complicated steps of culture in vitro and transplantation into a living body are essential, and advanced culture technology and its transplantation technology,
Culture equipment and time for culture are required.

[0004] In the treatment with pharmaceuticals, in recent years, with the expectation that an ointment (actosin ointment) containing dibutyl cyclic AMP, a derivative of cyclic AMP, will promote the proliferation of epidermal cells and fibroblasts. It has come to be applied to skin defect wounds. However, since epidermal cells proliferate only from the epidermis around the defective wound, it takes a considerable amount of time for epidermal cells that have extended from the normal skin to cover a wide range of skin defective wounds and become epidermis. There's a problem.

[0005]

DISCLOSURE OF THE INVENTION The present invention enables complete recovery in a short period of time without the need for advanced culture techniques and equipment, without the need for flap sampling or skin harvesting that places a heavy burden on patients. Therefore, an object of the present invention is to provide an artificial dermis useful for treating a wide range of full-thickness skin defect wounds.

[0006]

It has been known that epithelial cells called outer root sheath cells are abundantly attached to a removed hair follicle. This outer root sheath cell,
It is a cell that can differentiate into epidermal cells. The reason for the healing of the pericarp part of the I / II degree burn or the thin stratified skin graft is that the outer root sheath cells remaining on the damaged surface are differentiated and proliferate into epidermal cells and cover the entire damaged part. However, since the dermis component does not exist in the full-thickness skin defect wound, even if the self-extracted hair follicle (hair follicle extracted from the patient) is directly transplanted, the outer hair root is similar to the case of the cultured epidermal sheet transplantation described above. Sheath cells do not survive. Therefore, outer root sheath cells differentiate into epidermal cells.
It cannot be expected to proliferate, and in fact no such clinical reports are known.

As a result of intensive studies, the present inventor has been able to utilize the property of the outer root sheath cells of autologous extracted hair follicles to differentiate into epidermal cells by using a specific artificial dermis, and to obtain a full-thickness skin defect. The present inventors have found that early treatment of wounds can be realized and completed the present invention. Specifically, when the artificial dermis of the present invention is applied to a full-thickness skin defect wound and a self-extracted hair follicle is transplanted onto the artificial dermis, outer root sheath cells differentiate into epidermal cells, and the skin Reproduce.

That is, the present invention comprises a wound contact layer comprising a composite matrix of fibrous atelocollagen and a modified atelocollagen having a helix content of 0 to 80%, and a moisture permeation controlling layer, and at least a length penetrating the wound contact layer. Provided is an artificial dermis for transplanting a self-extracted hair follicle having 10 to 10000 slits having a length of 3 to 10 mm and a width of 10 to 5000 μm per 100 cm ² .

More specifically, the present invention comprises a wound contact layer comprising a composite matrix of fibrillated atelocollagen and a modified atelocollagen having a helix content of 0 to 80% and a moisture permeation controlling layer, which penetrates at least the wound contact layer. An artificial dermis having 10 to 10,000 slits per 100 cm ² having a length of 3 to 10 mm and a width of 10 to 5000 μm, and when affixed to a damaged skin, the capillaries and fibroblasts are passed through the slits. By invading into the wound contact layer and then transplanting the outer root sheath cells attached to the self-extracted hair follicle to the surface of the wound contact layer that is removed by removing the moisture permeation control layer, the skin tissue is engrafted / Provided is an artificial dermis for self-extracted hair follicle transplantation that can be proliferated.

[0010] The content of the modified atelocollagen in the composite matrix is 1 to the total content of the fibrotic atelocollagen and the modified atelocollagen.
Preferably it is 〜50% by weight.

It is preferable that the composite matrix is thermally dehydrated and crosslinked by heating at 50 to 180 ° C. for 1 to 24 hours under a vacuum of less than 1 Torr.

[0012] The artificial dermis is preferably used in two phases for treating a full-thickness skin defect wound.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The artificial dermis of the present invention comprises a wound contact layer and a moisture permeation controlling layer. The wound contact layer consists of a composite matrix of fibrillated atelocollagen and modified atelocollagen with a helix content of 0-80%.
The collagen used in the present invention is not particularly limited, but is preferably derived from mammals. The animal species and the derived tissue in mammals are not particularly limited. Collagen is common to mammals, and any collagen derived from mammals is biocompatible with the human body. A preferred example is collagen derived from bovine dermis. Atelocollagen is obtained by removing the telopeptide region, which is the main antigenic site, from collagen using an enzyme or the like. Specifically, there is a method in which collagen is treated with an acid or an alkali and then treated with pepsin. In the present invention, collagen excluding the antigenic site,
That is, atelocollagen is used to suppress the expression of antigenicity of collagen. In the present invention,
The obtained atelocollagen is used after fibrillation. The fibrillated atelocollagen is obtained by neutralizing atelocollagen with a phosphate-based buffer or the like, and arranges atelocollagen molecules having a triple-chain helical structure in the longitudinal direction so that the whole is directional. It was made. In the present invention, the fibrillated atelocollagen contributes to stabilization of the structure of the wound contact layer and improvement of physical properties.

The modified atelocollagen used in the present invention is an atelocollagen having a helix content of 0 to 80%. The atelocollagen similar to that used for the above-mentioned fibrotic atelocollagen is subjected to heat treatment, chemical treatment,
It can be obtained by physical processing or the like. The denatured atelocollagen is preferably denatured by heat treatment. In the heat treatment, for example, atelocollagen is swollen with water to form an atelocollagen aqueous solution,
This is done by heating to 0 ° C. Since thermal denaturation of atelocollagen occurs at around 37 ° C., it is preferable to heat it to 40 ° C. or more because denaturation can be surely performed. Heating to 90 ° C. or less is preferable because irreversible denaturation of atelocollagen does not occur. A more preferred heat treatment is to hold at 40-80 ° C for 20-90 minutes. Thereby, the helix content can be set to about 20 to 30% by weight in a preferable range described later. The atelocollagen molecule before denaturation has a triple helix structure, but a part of the triple helix structure collapses due to denaturation to form a random coil, and the molecules come to exist irregularly. Therefore, the degree of denaturation of the denatured atelocollagen can be shown by the content of the triple helix structure (helix content). The modified atelocollagen used in the present invention has a helical content of 0 to 80%, preferably 0 to 50%, more preferably 20 to 30%. When the content is within the above range, the affinity with the cells described below becomes favorable. The helical content can be measured with a circular dichroism or infrared spectrophotometer (Macromolecules, 7 [6] (1)
974) p. L. Gordon, I .; V. Yannas
et al. , P. 954; Collection of Polymer Papers, 41 [8]
(1984) Nakura, Hashimoto et al., P. 473). In the present invention, the denatured atelocollagen contributes to the affinity for cells described below.

The content of denatured atelocollagen in the composite matrix is preferably 1 to 50% by weight, more preferably 5 to 30% by weight, based on the total content of fibrous atelocollagen and denatured atelocollagen. , 10 to 20% by weight. When the content is within the above range, the structural stability of the wound contact layer and the affinity with the cells become suitable.

The wound contact layer has a collagen density of 5 to 50.
mg / cm ³ , preferably 15-35 mg / c
More preferably, it is m ³ . This is because within the above range, the strength of the wound contact layer is sufficient. The thickness of the wound contact layer is 1
It is preferably from 10 to 10 mm.

The wound contact layer is preferably obtained by placing a mixed solution of fibrillated atelocollagen and denatured atelocollagen in an appropriate container and freeze-drying to form a porous body. By forming in this manner, the density of the wound contact layer falls within the above range. It is preferable that the wound contact layer is made of a porous material because engraftment of outer root sheath cells during the transplantation of a self-extracted hair follicle, which will be described later, and proliferation of epidermal cells resulting from the differentiation are facilitated.

As a specific preferred example of the wound contact layer, a “short-time heat-dehydrated crosslinked fibrillar collagen + denatured collagen complex” (artificial organ, 18
[1] (1989) Konishi et al., "A New Type of Collagen Material That Reconstructs Self-Tissue", p. 155-158).

The moisture permeation controlling layer is a layer for controlling water in a wound when the artificial dermis of the present invention is applied to a wound surface. The moisture permeation control layer does not allow the exudate to accumulate on the wound surface and keeps the wound surface moist by performing appropriate water vapor transmission. In addition, leakage of the protein component in the exudate to the outside is prevented. Furthermore, invasion of bacteria and the like from the outside is prevented. The material constituting the moisture permeation controlling layer is not particularly limited as long as it is a non-toxic material, but is made of a synthetic polymer such as a silicone elastomer, a polyacrylate ester, a polymethacrylate ester, and a polyurethane elastomer (including porous polyurethane). And more preferably a silicone elastomer. The thickness of the moisture permeation control layer is preferably from 5 to 300 μm, more preferably from 50 to 300 μm. As the moisture permeation control layer, for example, a thickness of 50 to 30
Medical silicone of 0 μm is preferably used.

The artificial dermis of the present invention is formed by laminating the above-mentioned wound contact layer and a moisture permeation controlling layer. For example, a mixed solution of fibrillated atelocollagen and denatured atelocollagen is placed in an appropriate container, freeze-dried to form a porous body, and then dried before the thin film serving as a moisture permeation controlling layer prepared from a synthetic polymer solution is dried. On the thin film, dried until the thin film is cured, and finally heat-treated. In particular, it is preferable that the last heat treatment is performed at 50 to 180 ° C. for 1 to 24 hours under a vacuum of less than 1 Torr.
A more preferable degree of vacuum is less than 0.05 Torr, a more preferable processing temperature is 100 to 115 ° C., and a more preferable processing time is 2 to 8 hours. By this heat treatment, the composite matrix as the wound contact layer is thermally crosslinked, and the structure becomes more stable. In addition, the artificial dermis of the present invention in which the composite matrix is thermally crosslinked by the heat treatment exhibits excellent biocompatibility. As described above, cross-linking the composite matrix for the purpose of structural stability and the like is a preferred embodiment of the present invention, but such cross-linking needs to be performed within the scope of the present invention. That is, it is necessary to crosslink so that the biocompatibility of collagen is not lost.

The artificial dermis of the present invention comprises a wound contact layer and a moisture permeation controlling layer as described above, and has a length of at least 3 to 10 mm, preferably 4 to 7 mm, and a width of 10 to penetrate the wound contact layer. ~ 5000 μm, preferably 50-3000 μm slits per 100 cm ²
The artificial dermis has 0 to 10000, preferably 100 to 2000. As described later, the artificial dermis of the present invention has a slit that penetrates the wound contact layer so that blood vessels and the like can easily enter the inside of the artificial dermis, and the outer root sheath cells of the self-extracted hair follicle are removed from the artificial dermis. Can be engrafted. The length of the slit provided in the wound contact layer,
When the width and density are in the above ranges, engraftment of outer root sheath cells is optimally performed. Slits having different lengths and / or widths may be mixed. The arrangement of the slits is not particularly limited as long as the density is within the above range and is substantially uniform, and may be random or some regular arrangement. The slit may be provided at least in the wound contact layer. However, when the slit is provided after the wound contact layer and the moisture permeation controlling layer are laminated, FIGS.
If the slit communicates with both layers as shown in the above, the adhesion between the artificial dermis of the present invention and the skin damaged part is more effectively prevented by preventing the exudate from being stored, and the production is easy. preferable. When the slit is provided only in the wound contact layer, the moisture permeation controlling layer may be applied after the slit is formed in the wound contact layer. Further, the slit may not include a slit that has not reached the wound contact surface or a slit that is open on the side surface of the artificial skin. The method for providing the slit is not particularly limited, and a known method can be used. For example, a method using a “skin graft mesher” for making a slit in a skin piece for transplantation and the like can be mentioned. The slit is not limited to a linear slit, but may be a circular shape, an irregular shape, or the like, and may have various shapes. Their size and density are the same as for the linear one.

The method of using the artificial dermis of the present invention and its effects will be described with reference to examples. The artificial dermis of the present invention is applied so that the surface of the wound contact layer is in close contact with the mother's bed at the skin damaged part in the treatment of a full-thickness skin defect wound, a third degree burn, and the like.
After application, body fluid gradually penetrates into the wound contact layer from the wet mother bed, and capillaries and fibroblasts penetrate into the wound contact layer from the mother bed through the slit. After the capillaries and fibroblasts reach the vicinity of the contact surface with the water permeation control layer,
Preferably, after 3 to 10 days or more have passed since the application,
The moisture permeation control layer of the artificial dermis is peeled off, and a self-extracted hair follicle is transplanted on the wound contact layer, and further protected with a closing film or the like. The outer root sheath cells present in the self-extracted hair follicles easily engraft on the wound contact layer due to the presence of capillaries and fibroblasts, differentiate into epidermal cells, and the differentiated cells proliferate and epidermis Becomes Thus, the use of the artificial dermis of the present invention enables the skin to be regenerated in a short period of time. Unlike the proposal of Japanese Patent No. 2722838 described above, when the artificial dermis of the present invention is used, the artificial dermis is composed of a complex matrix of fibrillated atelocollagen and denatured atelocollagen having a helix content of 0 to 80%, preferably fibrosis. It is obtained by freeze-drying a mixed solution of atelocollagen and denatured atelocollagen to form a porous body, and heating at 50 to 180 ° C. for 1 to 24 hours under a vacuum of less than 1 Torr, and having a slit of an appropriate size. On the surface of the wound contact layer, outer root sheath cells differentiate and epidermal cells proliferate. This is facilitated by the penetration of bodily fluids into the wound contact layer and the capillary and fibroblasts present in the mother bed reaching the vicinity of the surface of the wound contact layer through slits in the wound contact layer. . Also, unlike the proposal of Japanese Patent No. 2722838, in v
Since all steps are performed in vivo, there is a great advantage that a complicated step of culturing and transplanting can be omitted.

[0023] The artificial dermis of the present invention has good biocompatibility and adhesion to a skin damaged part in a full-thickness skin defect wound or the like because the wound contact layer is composed of a composite matrix of fibrillated atelocollagen and denatured atelocollagen. . In addition, the artificial dermis of the present invention does not have foreign matter or inflammatory (inflammation) in the components of the wound contact layer and the moisture permeation control layer, which inhibit the engraftment, differentiation, and proliferation of outer root sheath cells. Further, the artificial dermis of the present invention has slits of an appropriate size in the wound contact layer at an appropriate density, so that capillaries and fibroblasts that promote engraftment, differentiation and proliferation of outer root sheath cells are extremely internal. Easy to invade. And since the artificial dermis of the present invention has a moisture permeation control layer, until the self-extracted hair follicle is transplanted,
External infection can be prevented and the wound contact layer can be kept in a moderately moist environment. For these reasons, the effect of skin regeneration by the artificial dermis of the present invention is exhibited.

Further, the artificial dermis of the present invention is transplanted in two phases as described above, that is, the transplantation is carried out by shifting the timing of application to the damaged skin and the timing of transplantation of the auto-extracted hair follicle. Is preferred. This is to allow time for capillaries and the like to enter the wound contact layer. On the other hand, the artificial dermis of the present invention can also be used by transplanting it temporarily. That is, it is also possible to remove the moisture permeation control layer and attach the self-extracted hair follicle simultaneously with the application to the skin damaged part. In this case, the engraftment rate of the outer root sheath cells is somewhat inferior to the case of two-stage transplantation, but there is an advantage that the burden on the patient is small.

As described above, even if a self-extracted hair follicle is directly transplanted into a full-thickness skin defect wound that also lacks dermis components, the outer root sheath cells do not survive and the skin is not regenerated. This is considered to be because the engraftment, differentiation, and proliferation of outer epithelial cells called “epidermal cells” require the dermis called “mesenchymal” or a dermis-like tissue equivalent thereto.

When the artificial dermis of the present invention is applied to a damaged skin, capillaries and fibroblasts quickly enter the wound contact layer, and the capillary blood vessels and the wound contact layer are integrated. The dermis-like tissue forms very early. It is considered that the artificial dermis of the present invention enables engraftment, differentiation, and proliferation of outer root sheath cells to form a dermis-like tissue in this manner. That is, the artificial dermis of the present invention, by adopting the above-described configuration, allows capillary blood vessels and fibroblasts to penetrate into the wound contact layer through the slit, and then removes the moisture permeation control layer to form the wound contact layer. Transplant the outer root sheath cells attached to the self-extracted hair follicle to the surface to engraft epithelial tissue.
An artificial dermis that can grow.

[0027]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. (Example 1) (1) Preparation of mixed solution of fibrillated atelocollagen-denatured atelocollagen 1 g of bovine atelocollagen (manufactured by Koken Co., Ltd.) was dissolved in dilute hydrochloric acid of pH 3.0 at a temperature of 4 ° C to 0.3 (W). /
V) A hydrochloric acid solution was prepared. The solution is filtered and sterilized by passing it through two filters having pores of 0.8 μm and 0.2 μm in diameter, and then stirred while maintaining at 4 ° C. so that the final pH becomes 8. An acid buffer was added to obtain an atelocollagen solution having a final concentration of 0.27 (W / V)%. Then, after immersing in a thermostat at 37 ° C. for 4 hours to obtain a fibrillated atelocollagen (FC) solution, the solution was concentrated by a centrifugal operation under aseptic conditions to obtain a concentration of 2.5 (W / V).
% FC solution was obtained. On the other hand, a 0.3 (W / V)% hydrochloric acid solution of bovine atelocollagen after the above-mentioned filtration sterilization is freeze-dried, and redissolved again in sterile distilled water to 2.5 (W / V)%. This was left in a thermostat at 60 ° C. for 30 minutes to cause thermal denaturation, and the denatured atelocollagen (H
AC) solution. The helical content of the modified atelocollagen was about 34%. 2.5 (W /
V)% HAC solution and concentration 2.5 (W / V)% FC
The solution was mixed such that HAC / (FC + HAC) = 0.1, stirred well, and fibrotic atelocollagen-
A mixed solution of denatured atelocollagen was obtained.

(2) Freeze-drying The mixed solution of fibrillated atelocollagen-denatured atelocollagen obtained in (1) was poured into a plastic container having a predetermined shape, rapidly cooled to -30 ° C or lower, and sufficiently frozen. Thereafter, the mixture was freeze-dried at -40 ° C under a vacuum of less than 1 Torr to obtain a sponge-like composite matrix composed of fibrous atelocollagen-denatured atelocollagen.

(3) Affixing of silicone film Medical silicone (manufactured by Dow Corning) on Teflon
Was applied using a precision coating tool (applicator).
The sponge-like composite matrix obtained in (2) was placed on the applied film and left at 55 ° C. for 3 hours or more.

(4) Introducing a slit The sponge-like composite matrix with a silicone film obtained in (3) was slit with a cutter having a magnification of 1.5 times using a skin graft mesher (manufactured by Zimmer). . After the introduction of the slit, the sponge-like composite matrix with the silicone film had about 1660 slits having a length of 4 mm and a width of 20 μm per 100 cm ² .

(5) Thermal Dehydration Crosslinking The sponge-like composite matrix with a slit provided with the silicone film obtained in (4) was heated to 110 ° C. under a vacuum of less than 0.05 Torr and maintained for 2 hours to perform thermal dehydration crosslinking. . Thereafter, the temperature was returned to normal temperature and normal pressure to obtain the artificial dermis of the present invention.

Comparative Example 1 <Equivalent to Example 1 without Slit> An artificial dermis without a slit was obtained in the same manner as in Example 1 except that the step (4) was omitted.

(Comparative Example 2) <Glutaraldehyde cross-linked, without HAC, with slit> The FC solution of Example 1 (1) having a concentration of 2.5 (W / V)% was frozen by the same method (2). After drying and attaching the silicone film by the method of (3), a slit was introduced by the method of (4). The obtained fibrous atelocollagen matrix with a slit having a silicone film consisting of only fibrous atelocollagen is heated to 110 ° C. under a vacuum of less than 0.05 Torr
, And maintained for 24 hours for thermal dehydration crosslinking. At normal temperature,
After the pressure was returned to normal pressure, it was aseptically immersed in a 0.05% glutaraldehyde solution for 2 hours to further highly crosslink. Thereafter, the resultant was washed with sterile distilled water to remove glutaraldehyde as a crosslinking agent.

(Comparative Example 3) <Glutaraldehyde cross-linked, HAC-equipped, with slit> Fibrous atelocollagen modified by the method of (1), (2), (3) and (4) in Example 1 A sponge-like composite matrix having a slit with a silicone film and made of atelocollagen was crosslinked in the same manner as in Comparative Example 2.
That is, it is heated to 110 ° C. under a vacuum of less than 0.05 Torr and maintained for 24 hours, thermally dehydrated and crosslinked, returned to normal temperature and normal pressure, and then aseptically immersed in a 0.05% glutaraldehyde solution for 2 hours. By doing so, it was more highly crosslinked.
Thereafter, the resultant was washed with sterile distilled water to remove glutaraldehyde as a crosslinking agent.

(Comparative Example 4) <Equivalent to Example 1 without HAC (with slits)> The FC solution having a concentration of 2.5 (W / V)% of Example 1 (1) was obtained by the same method (2). After freeze-drying and attaching a silicone film by the method of (3), a slit is introduced by the method of (4), and the obtained fibrous atelocollagen matrix with a silicone film and consisting of only the fibrotic atelocollagen is used. Thermal dehydration crosslinking was carried out by the method (5).

Next, the artificial dermis of the present invention obtained in Example 1 was applied to a full-thickness skin defect wound prepared on the back of a rat, and its suitability as an artificial dermis was evaluated. As a comparative control, the artificial dermis of Comparative Examples 1 to 4 were similarly evaluated.

Test Example 1 (1) Attachment of Artificial Dermis A Sprague-Dawley female rat (10 weeks old) was shaved on the back under Nembutal anesthesia and then 2 ×
A full thickness skin defect wound with a 2 cm panniculus carnosus wound was created. The artificial dermis of any of Example 1 or Comparative Examples 1 to 4 was stuck thereon (all in such a manner that the wound contact layer side, that is, the surface of the sponge-like matrix without a silicone film was in contact with the mother bed). Affixed). A sterile gauze and a transparent polyurethane occlusive film (trade name: Bioc) are placed on the artificial dermis.
plus, manufactured by Johnson & Johnson)
, And pressed and fixed with an elastic bandage.

(2) Transplantation of self-extracted hair follicles One week after the artificial dermis was applied, 10 hairs of the whiskers were removed from the test rats, and only the hair follicles at the roots of the hair were cut off. For 30 seconds, and then washed thoroughly with sterilized saline. Remove the elastic bandage, polyurethane closing film, sterile gauze and silicone membrane, and on the exposed collagen matrix layer,
The obtained self-extracted whisker follicle was seeded so as to be randomly dispersed. From above, a 2 × 2 cm polyurethane closing film (trade name: Bioclive)
, And pressed and fixed with an elastic bandage. As a control, a group in which only the artificial dermis of Example 1 was applied and no hair follicle was transplanted, and a group in which the artificial dermis was not applied and only the hair follicle was transplanted were provided.

(3) Observation of progress Two weeks after application (one week after transplantation of hair follicles), the state of the wound was visually observed, the animal was sacrificed and the transplant was examined histopathologically. . 3 weeks after application (2 weeks after hair follicle transplantation)
Weeks), the animals to be sacrificed were exchanged for the closing film and the elastic bandage two weeks after the application (one week after the hair follicle transplantation). Three weeks after application (two weeks after hair follicle implantation), the state of the wound was visually observed, the animal was sacrificed and the transplant was examined histopathologically.

The results of gross findings are shown in Table 1 and the results of histopathological examination are shown in Table 2. Artificial dermis of the present invention (Example 1)
It can be seen that the epidermis regenerates very early when a self-extracted hair follicle is transplanted using the method. FIG. 3 is a photomicrograph at about 180 × magnification of the artificial dermis 2 weeks after application (1 week after hair follicle transplantation) when the artificial dermis of Example 1 was used to implant a self-extracted hair follicle. Many fibroblasts and capillaries (→) infiltrate into the wound contact layer to form a dermis-like tissue (*). Outer root sheath cells of the transplanted hair follicle (★) engraft on the surface of the dermis-like tissue or inside the dermis-like tissue.
Proliferate, and a reconstructed epidermis on the islets (島) is observed on the dermis-like tissue.

On the other hand, when the artificial dermis of Comparative Example 1 having no slit is used, the transplanted outer root sheath cells survive but do not proliferate. This is because, since there is no slit, blood vessel invasion into the wound contact layer is delayed as compared with Example 1. Since the epidermis regeneration from the surrounding normal skin is only slightly recognized, the epidermis is not completed even after three weeks. When the artificial dermis of Comparative Example 2 was used, the outer root sheath cells of the transplanted hair follicle did not engraft. This is because the cross-linking method is not appropriate, the biocompatibility of collagen itself is lost,
Conversely, the result is a foreign body reaction and an inflammatory reaction, which indicates that the conditions for collagen cross-linking are extremely important factors. Since the epidermis regeneration from the surrounding normal skin is only slightly recognized, the epidermis is not completed even after three weeks. Even when the artificial dermis of Comparative Example 3 was used, the same results as in Comparative Example 2 were obtained. This is also not a suitable crosslinking method. When the artificial dermis of Comparative Example 4 was used, the outer root sheath cells of the transplanted hair follicles survived but did not proliferate. Although the crosslinking method is appropriate, it does not contain heat-denatured collagen, and as a result, cell affinity (biocompatibility) in the wound contact layer is reduced, and neutrophil infiltration is promoted. And because epidermis regeneration from surrounding normal skin is only slightly recognized,
Epidermalization is not complete after 3 weeks. Even when the artificial dermis of Example 1 was used, when the auto-extracted hair follicle was not transplanted, epidermal regeneration from surrounding normal skin was only slightly recognized, so that epidermis formation was not completed even after 3 weeks. . Further, when only the self-extracted hair follicle was directly transplanted to the damaged area of the skin without using the artificial dermis of Example 1, the outer root sheath cells did not survive,
Epidermis formation is not complete even after three weeks, since only a slight regeneration of the epidermis from the surrounding normal skin is observed.

[0042]

[Table 1]

[0043]

[Table 2]

[0044]

The artificial dermis according to the present invention has excellent biocompatibility and stability in the living body itself and also has a suitable size slit at a suitable density, so that early cell invasion from the mother bed can be achieved. Excellent in properties. With the artificial dermis of the present invention,
Successful autologous follicle transplantation, which no one could have imagined or achieved, could reconstruct the skin with extensive full-thickness skin defect wounds with little burden on the patient.

[Brief description of the drawings]

FIG. 1 is a plan view of one embodiment of the artificial dermis of the present invention.

FIG. 2 is a perspective view of one embodiment of the artificial dermis of the present invention.

FIG. 3 is a micrograph showing that the epidermis is regenerated by the artificial dermis of the present invention (about 180 times).

[Explanation of symbols]

 1 wound contact layer 2 moisture permeation control layer 3 slit

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Ken Uematsu 1500 Inoguchi, Nakai-machi, Ashigara-gun, Kanagawa Prefecture Terumo F-term (reference) 4C081 AA02 AA12 AB19 AB20 BA12 BB01 CA082 CA212 CA272 CB042 CB052 CC04 CC06 CD131 CD34 DA02 DA12 DC02 DC03 DC06

Claims (4)

[Claims] 1. A wound contact layer comprising a composite matrix of fibrillated atelocollagen and a modified atelocollagen having a helix content of 0 to 80%, and a moisture permeation regulating layer, having a length of at least 3 to 10 penetrating the wound contact layer.
mm, the slit width 10~5000μm 100cm ²
An artificial dermis for transplanting a self-extracted hair follicle having 10 to 10000 pieces per hair. 2. The content of the modified atelocollagen in the composite matrix is 1 to the total content of the fibrotic atelocollagen and the modified atelocollagen.
The artificial dermis according to claim 1, wherein the amount is from 50 to 50% by weight. 3. The artificial dermis according to claim 1, wherein the composite matrix is heat-dehydrated and crosslinked by heating at 50 to 180 ° C. for 1 to 24 hours under a vacuum of less than 1 Torr. 4. The artificial dermis according to any one of claims 1 to 3, which is used for two-stage treatment of a full-thickness skin defect wound.