JPH1080438A - Tissue-containing artificial dermis and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to close a wound by forming the epidermis and dermis tissues with one time of epidermization in the treatment of a full- thickness dermal defect wound damaged down to the dermis tissues by a wound, thermal burn, etc. SOLUTION: This artificial dermis for temporarily treating the full-thickness dermal defect wound is formed by laminating a silicone layer 3 on one surface of a collagen sponge layer 2 having recessed parts or hole parts 1 for contg. the dermal tissues including the epidermis and dermis tissues. In such a case, the artificial dermis contains a shin tissues 4 contg. the epidermis and dermis tissues in order of the epidermis tissues 5 and the dermis tissues 6 from he silicone layer 3 side in these recessed parts or hole parts 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel artificial dermis and a method for producing the same. More specifically, the present invention relates to an artificial skin that can close the wound by forming the epidermis and the dermis tissue by a single skin grafting in the treatment of a full-thickness skin defect wound that has been damaged to the dermis tissue by a wound or a burn. The present invention relates to the dermis and a method for producing the dermis.

[0002]

2. Description of the Related Art Conventionally, in treating a patient whose dermis tissue has been significantly damaged by diseases or wounds such as burns, cutaneous wounds, traumatic skin defect wounds, pressure ulcers, etc., a necrotic tissue is removed to remove a full thickness dermal defect wound. Autologous skin grafting has been practiced in which separated skin is collected from healthy skin and transplanted. However, if the area of damage is large, it is not possible to cover all the defect sites in all layers with a single autograft.
A third autologous graft is performed in a timely manner.

[0003] However, autogenous skin grafting has a problem that it cannot cope with a case where a required amount of separated skin cannot be ensured. For example, the application of cultured skin and artificial dermis has attracted attention as a new medical technology that can be used instead.

[0004] However, the cultured skin has an extremely imperfect structure as compared with the self-skinned skin graft, and the survival rate is greatly affected by the infection of the wound surface. In addition, the cell culture requires time and equipment, and is enormous. It has various drawbacks, such as high cost, and has not been put to practical use.

On the other hand, various artificial dermis have been developed and their usefulness has been confirmed clinically (British J. Pla.
st. Surg., 43, 47-54 (1990)). However, when the wound area is large in the conventional artificial dermis, the dermis-like tissue is constructed but the epidermal tissue cannot be regenerated. Therefore, after the construction of the dermis-like tissue, the second operation for constructing the epidermal tissue, ie, the second operation, Secondary skin grafting was essential. For this reason, the physical and mental distress of the patient was great, and the economic burden due to prolonged hospitalization and the like was great.

[0006]

SUMMARY OF THE INVENTION In order to make up for the above-mentioned drawbacks of the conventional artificial dermis, the present invention has been proposed to speed up wound closure by forming dermis-like tissue and epidermal tissue.
An object of the present invention is to provide an artificial dermis that does not require a second skin graft operation. Another object of the present invention is to provide a method for producing the artificial dermis.

[0007]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in view of the above-mentioned problems of the prior art, and as a result, a collagen layer has been laminated on a collagen sponge layer, and the collagen sponge layer has a concave portion or a hole portion. By using an artificial dermis in which a subject (patient) own or a skin fragment immunologically similar to the subject (patient) is incorporated into a bilayer artificial dermis having a specific structure having Even if is relatively widespread, immediately after application, skin tissue cells are regenerated and invaded not only from the wound surface and wound margin but also from the skin fragments so that skin tissue can be reconstructed at an early stage. By using a skin fragment having an epidermis and dermis tissue as an artificial dermis to be incorporated into the artificial dermis, the skin can be naturally and quickly superimposed on the dermis tissue without implanting the epidermis tissue by two operations. Wounds by tissue forms is found that closed early, thereby completing the present invention.

[0008] That is, the present invention relates to an artificial dermis for treating a full-thickness skin defect wound, wherein the collagen sponge layer has a concave portion or a pore portion for accommodating skin tissue including epidermis and dermis tissue. The present invention provides an artificial dermis having a silicone layer laminated on one side.

The present invention also relates to an artificial dermis comprising a collagen layer laminated on one side of a collagen sponge layer having a concave portion or a hole, wherein an epidermis tissue or a dermis is formed in the concave portion or the hole from the silicone layer side. Provided is a tissue-containing artificial dermis comprising skin tissues including epidermis and dermis tissues in the order of tissues.

[0010] The present invention further provides a full-thickness skin defect wound of an artificial dermis in which a silicone layer is laminated on one surface of a collagen sponge layer having a concave portion or a hole portion for accommodating skin tissue including dermis and epidermal tissue. For the manufacture of a tissue-containing artificial dermis which is to be therapeutically treated.

Further, the present invention provides a step of placing a skin tissue including an epidermis and a dermis tissue into a recess or a hole of an artificial dermis obtained by laminating a silicone layer on one surface of a collagen sponge layer having a recess or a hole. The present invention also relates to a method for producing the above-mentioned artificial dermis containing tissue.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The artificial dermis according to the present invention is an artificial dermis that treats a full-thickness skin defect wound temporarily, and as shown in FIG. 1, for example, a skin containing epidermis and dermis tissue A collagen layer (reference numeral 3) is laminated on one surface of a collagen sponge layer (reference numeral 2) having a concave portion or a hole portion (reference numeral 1) for receiving a tissue.

In the present invention, “full-thickness skin defect wound”
It refers to a wound in which not only epidermal tissue but also dermal tissue is lost or lost by removing diseases or wounds such as burns, skin wounds, traumatic skin defect wounds, pressure ulcers, and necrotic tissue. However, the range of the wound is not particularly limited, and encompasses not only those in which the full-thickness skin tissue is lost or deficient over a wide range, but also wounds in which some full-thickness skin tissue is lost or deficient. .

[0014] In the present invention, "temporarily" means a second time,
It is used in the meaning that only one skin grafting is performed without performing a third skin grafting treatment or a split skin grafting treatment, and “treating” means that a full-thickness skin defect wound is healed / improved. Means that the dermal tissue is formed and the wound is closed by the epidermal tissue being stretched over the damaged dermis.

Therefore, the artificial dermis of the present invention is particularly useful when applied to a full-thickness skin defect wound in which it is difficult to regenerate the dermis and epidermal tissue.

The collagen sponge layer (FIG. 1, reference numeral 2) used in the present invention is a sponge which has a certain strength by physically or chemically treating collagen, which is a highly biocompatible water-containing polymer. What was made into the shape or the foam shape is used.

In general, collagen itself is a material derived from a living body, so it is a suitable material having high affinity for cells and tissues and excellent biocompatibility. On the other hand, collagen is easily decomposed and absorbed in a living body. Therefore, a device for strengthening the physical properties is required.

From this point of view, the collagen sponge of the present invention does not decompose until a dermis-like tissue is formed by introducing chemical cross-linking or the like, and is replaced with the dermis-like tissue in about 2 to 3 weeks after skin grafting. Designed to. For example, for this purpose, a method of thermally dehydrating and crosslinking collagen sponge, or a method of chemically crosslinking with glutaraldehyde, hexamethylene diisocyanate, epoxy, etc., may be mentioned, but preferably, at about 105 ° C. under vacuum. About 2
Intramolecular cross-linking by treatment for 4 hours, followed by 0.2
% Glutaraldehyde solution (0.05N acetic acid)
This is a method of performing intermolecular cross-linking by immersion for about 24 hours.

The density of collagen sponge is as follows:
Although not particularly limited, 0.001 to 0.1 g / c
m ^3, preferably 0.003~0.03g / cm ^3, more preferably from 0.005~0.015g / cm ^3, it is more desirable that among them 0.01 g / cm ³ before and after. However, it may be in the range of 0.005 to less than 0.01 g / cm ³ .

The collagen used is preferably atelocollagen from which the antigenic determinant has been removed by an enzyme in terms of suppressing the expression of antigenicity.

The average diameter of the foam of the foam structure of the collagen sponge is 10 to 500 μm, preferably 50 to 300 μm.
μm is desirable. By having such a foamed structure, the invasion of fibroblasts can be facilitated and the formation of granulation tissue can be improved, and at the same time, excretion of exudate and blood derived from a living body can be facilitated to reduce the residual body fluid and the like. It is possible to enhance the adhesion to the artificial dermis of the present invention.

The thickness of the sponge layer is 0.5 to 10 mm, preferably 1 to 7 mm, and more preferably 2 to 4 mm in consideration of the thickness of the dermis layer and the thickness of the tissue fragment to be contained.

The artificial dermis of the present invention has a basic structure in which a silicone layer (reference numeral 3) is laminated on one surface of the above-mentioned collagen sponge layer.

[0024] The silicone layer is intended to suppress insufficiency and prevent infection, and is finally removed after the dermis and epidermis are formed under the silicone layer.

The silicone layer used in the present invention has a water vapor permeability of usually 0.1 to 100 mg / cm ² / h, preferably 0.5 to ¹⁰ mg / cm ² / h, more preferably 1 to 5 mg / cm ^2. / H is not particularly limited as long as it is within the range of / h. Further, any material other than silicone may be used as long as it has such water vapor permeability.

The artificial dermis of the present invention is characterized in that, in the above-mentioned two-layer structure, the collagen sponge layer has a concave portion or a hole exemplified by reference numeral 1 in FIG. The recess or hole is formed for the purpose of inserting skin tissue including epidermis and dermis tissue therein, and is used for that purpose. Therefore, the shape of the concave portion or the hole, the depth, the size, the hole diameter, and the like of the concave portion are not particularly limited as long as the object can be achieved.

In the present invention, the term “recess” means a hole, dent or depression formed without penetrating the collagen sponge layer.
The depth of the skin is not particularly limited, but is usually such that the skin tissue including the epidermis and the dermis tissue enters, and a depth less than the thickness of the collagen sponge layer, specifically, a depth of about 2 to 4 mm. Can be On the other hand, the hole means a hole penetrating at least the collagen sponge layer. Such pores may be formed continuously in two layers of a collagen sponge layer and a silicone layer, but from the viewpoint of preventing various bacterial infections of the diseased part during the period of application of the artificial dermis of the present invention,
Preferably, the holes do not penetrate the silicone layer.

In a preferred embodiment of the present invention, the diameter of the recess or the hole is not particularly limited, but is preferably in the range of 0.1 to 10 mm, more preferably 1 to 5 mm.

The pore diameter and the number of the recesses or pores formed in the artificial dermis correspond to the size and the number of the skin tissue to be put therein, and the larger the diameter (or the larger the number), the larger the diameter. ) Although wound closure (treatment) is quicker, on the other hand, the amount of healthy skin tissue to be collected increases, and damage to healthy tissue is inevitable. Therefore, the diameter and number of the recesses or holes formed in the artificial dermis can be appropriately adjusted based on the relationship between the speed of wound closure and the collection of healthy skin.

In general, the smaller the ratio of the area of the entire artificial dermis to the total pore area of the concave portion or hole into which the artificial dermis is inserted (hereinafter referred to as “enlargement ratio”), the sooner the wound is closed. From, usually in the range of 5 to 100 times,
Preferably, a magnification of 10 to 50 times is exemplified.

The artificial dermis of the present invention described above is used by incorporating a skin tissue containing epidermis and dermis cells into the depressions or pores of the collagen sponge layer. The skin tissue to be used is not particularly limited as long as the immunogenicity of the subject to whom the artificial dermis is applied is similar or the same, and immunological rejection does not occur by transplantation. And more preferably the subject's own healthy skin tissue to which the artificial dermis is applied. The skin tissue is required to contain at least an epidermal tissue and a dermal tissue.

For example, as in the present invention, a two-layer coating material comprising a collagen sponge layer and a silicone layer,
When a non-skin tissue is applied to a full-thickness skin defect wound, fibroblasts and capillaries invade from the wound surface and its periphery, fill the pores of the sponge with its own collagen tissue, and finally Regenerate the dermis-like tissue. However, when the wound area is large, the epidermis layer does not regenerate, so it is necessary to close the dermis layer by thin skin grafting on this (this is called secondary skin grafting).

However, in the artificial dermis of the present invention in which the bilayer artificial dermis is provided with a recess or a hole, the skin tissue including the epidermis and the dermis tissue is put into the hole, and is applied to the full-thickness skin defect wound.
When transplanted, skin cells proliferate immediately from the incorporated skin tissue, so that the construction of the dermal tissue can be achieved at an early stage, and the skin tissue incorporated into the artificial dermis contains epidermal cells Therefore, the epidermal tissue extends from there, and the wound is closed not only from around the wound but also from several places at the center of the wound.

Therefore, according to the present invention, even in the case of a full-thickness skin defect wound or the like in which the wound area is large and the conventional method requires secondary skin grafting, the wound is wound early by a single skin grafting. Has the advantage of closing and healing.

The tissue to be used is, from the viewpoint of simplicity, etc.,
It is preferable to use the fragments of the healthy skin of the subject collected with a skin trepan or the like for tissue collection as it is, but it is necessary to perform some treatment such as antibacterial treatment to prevent infection by bacteria and increase the survival rate. Can also. In addition, a bilayer artificial dermis for containing a tissue fragment may be prepared in advance using an antibacterial material, or the artificial dermis may be subjected to an antibacterial treatment or the like before the tissue fragment is placed.

The skin tissue collected from the healthy skin is placed in the concave portion or hole of the artificial dermis in the order of the epidermal tissue and the dermal tissue so that the epidermal tissue comes to the silicone layer side.

The present invention also provides the above-mentioned artificial dermis, wherein the collagen sponge layer contains skin tissue in a concave portion or a hole portion thereof.

As described above, the skin tissue used is preferably one having immunogenicity similar or identical to the subject to which the artificial dermis is applied, and containing at least epidermal and dermal tissues.

In the tissue-containing artificial dermis of the present invention, as shown in FIG. 2, such a skin tissue (reference numeral 4) is formed in a recess or a hole (reference numeral 1) formed in a collagen sponge layer (reference numeral 2). (Reference numeral 5), dermal tissue (reference numeral 6)
Are arranged in such a manner that the epidermal tissue comes to the silicone layer side (reference numeral 3).

The method for producing such a tissue-containing artificial dermis is not particularly limited. Usually, an artificial dermis comprising a collagen sponge layer having a concave portion or a hole and a silicone layer laminated on one surface thereof is prepared. It is manufactured by putting skin tissue including epidermis and dermis tissue into the concave portion or hole.

The artificial dermis obtained by laminating a silicone layer on one side of a collagen sponge layer having a concave portion or a hole is not particularly limited by the method for producing the same. For example, after preparing a two-layer artificial dermis composed of a collagen sponge layer and a silicone layer, a concave portion or a hole may be formed in the collagen sponge layer, or a collagen sponge layer having a concave portion or a hole may be formed. Alternatively, it may be manufactured by laminating a silicone layer on one side so as not to block the hole.

In the bilayer artificial dermis having a recess or a hole obtained in this way, the skin or tissue having immunogenicity similar or identical to the subject to which the artificial dermis is applied is applied to the recess or the hole. Then, a substance containing at least the epidermis and the dermis is placed in the order of the epidermis and the dermis so that the epidermis comes on the silicone layer side.

[0043]

According to the artificial dermis of the present invention, even in the case of a wound (a full-thickness skin defect wound) in which dermal tissue is lost and regeneration is difficult, the artificial dermis can be used without a second skin graft. It can be epithelialized early. Therefore, by using the artificial dermis of the present invention, it is possible to treat a full-thickness skin defect wound easily and with a reduced mental and physical burden on a patient.

Further, since the present invention requires only a small amount of skin tissue as a healthy skin tissue to be collected, the skin can be easily harvested even from a narrow range or a bent portion, and the influence on the healthy skin portion is small. I'm done. It is also useful when there are many skin-cutting parts such as burns over a wide area. Furthermore, unlike the conventional artificial dermis, the artificial dermis of the present invention does not require cell culture, so it can be transplanted in a short period of time, has little concern about bacterial infection, and can be widely applied to burn treatment in general facilities. It is practical.

[0045]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited to these examples.

Example 1 (1) Preparation of artificial dermis Atelocollagen (manufactured by Nitta Gelatin Co., Ltd.) prepared by enzymatically treating type I collagen derived from pig tendon was adjusted to pH 3 with dilute hydrochloric acid. This was stirred at high speed with a homogenizer (10,0
(00 rpm, 10 minutes), immediately poured into a stainless steel container, and frozen at -40 ° C. This was further freeze-dried under vacuum to produce a collagen sponge. Next, this collagen sponge was insolubilized by introducing thermal dehydration crosslinking at 105 ° C. under vacuum (0.01 torr. Or less) for 24 hours. The obtained collagen sponge was stuck on a silicone thin film formed on a Teflon flat plate and dried sufficiently. The two-layer material obtained in this manner was mixed with a 0.2% glutaraldehyde solution (0.05 N).
(Acetic acid solution) (24 hours, 5 ° C.) to introduce chemical crosslinking. This was sufficiently washed with ion-exchanged water to remove the cross-linking agent, and then the washing solution was replaced with a 15% ethanol solution. Then, the solution was rapidly frozen at -135 ° C and further freeze-dried in vacuum.

The obtained two-layered material is (30 mm × 30 m
m) Nine holes for inserting skin tissue at intervals of about 8 mm in the collagen sponge layer using a 3 mm diameter skin trepan (manufactured by Stiefel Laboratorium) so as not to damage the silicone layer. (Magnification 14 times: (3 × 3) ÷ (0.15 × 0.15 × 3.1)
4 × 9) = 14).

(2) Preparation of Tissue-Containing Artificial Dermis On the other hand, using the same skin trepan, 9 pieces of whole-layer skin including epidermis and dermis were collected from healthy skin,
The tissue-containing artificial dermis of the present invention was prepared by placing the epidermis side in contact with the silicon sheet side in the collagen sponge layer of the artificial dermis prepared in advance with holes.

[0049]

【test】

Transplantation Test 1 The tissue-containing artificial dermis obtained in Example 1 was transplanted to the white skin on the back of a guinea pig (inbred guinea pig, Strain 2 Slc, Japan SLC, weight about 300 g), and the wound was healed. I saw In addition, the black skin part of the guinea pig to be transplanted was used as the skin incorporated into the artificial dermis.

First, a guinea pig was dehaired under Nembutal anesthesia, and a 30 × 30 mm full-thickness skin defect wound in which the fat layer was completely exposed on the back white skin of the guinea pig sanitized with isodine was prepared, followed by hemostasis and drying. The tissue-containing artificial dermis having the black skin tissue incorporated therein was transplanted to the artificial dermis prepared as described above, and the periphery was sutured with a nylon thread, followed by gauze application and tie-over fixation. The tie-over fixation is a fixing method in which a gauze is placed on a treatment surface, and a suture sewn around the gauze is ligated to a suture on the opposite side so as to pass over the gauze.

One week after transplantation, the transplant site was excised and
A tissue specimen was prepared by fixing with 0% formalin, and stained with HE (hematoxylin / eosin) to examine the engraftment state of the tissue-containing artificial dermis. As a result, the healthy black skin, which was collected with the skin trepan and transplanted and incorporated into the bilayer artificial dermis, survived including the hair root, and no inflammation was observed in that part.

In addition, the presence or absence of accumulation of exudate under the artificial dermis was observed. The exudate did not accumulate, and the adhesion between the artificial dermis and the wound site was good.

Three weeks after the transplantation, the newly regenerated collagen tissue is filled in the space of the collagen sponge,
The granulation tissue appeared complete and the collagen sponge appeared to have disappeared. It was also confirmed that epithelialization progressed from the epidermis portion of the skin transplanted and incorporated into the bilayer artificial dermis, and that the transplanted black skin survived.

One month after transplantation, although slightly thinner than normal skin, the upper layer of the epidermis was sufficiently keratinized, and epidermis formation and skin reconstruction were almost completed.

After a further long period of time, it was confirmed that the transplanted black skin completely survived, and that black hair was elongated.

After the transplantation, no wound contraction on the transplanted surface was observed.

Transplantation Test 2 The size of the artificial dermis was increased from 30 mm to 50 mm on a side, and the tissue-containing artificial dermis including nine skins collected with a skin trepan having a diameter of 3 mm (magnification 39 times, FIG. 3 (a)) Tissue-containing artificial dermis containing 25 skins (magnification 14x, FIG. 3)
(B)) and, for comparison, guinea pigs were transplanted using the two-layer artificial dermis containing no skin tissue (FIG. 3 (c)) in the same manner as in the transplantation test 1 and compared. .

When an artificial dermis including 25 skins was implanted, it was confirmed that the epithelium had formed after about 2 weeks.
When an artificial dermis including nine skins was implanted, epithelialization occurred about 3 weeks later. On the other hand, when a two-layer artificial dermis containing no skin was used, formation of granulation tissue was confirmed in about 2 weeks, but it took 4 weeks or more for epithelialization.

[Brief description of the drawings]

FIG. 1 is a plan view (a) and a cross-sectional view (b) of a bilayer artificial dermis of the present invention having a recess or a hole in a collagen sponge layer into which a skin tissue is inserted, as viewed from the collagen sponge layer side.
Is shown.

[FIG. 2] In a concave portion or a hole portion of a collagen sponge layer,
FIG. 2 shows a plan view (a) and a cross-sectional view (b) of the tissue-containing artificial dermis of the present invention containing at least the skin tissue including the epidermis and the dermis tissue as viewed from the collagen sponge layer side.

FIG. 3 shows a plan view of a 50 mm × 50 mm artificial dermis used in transplantation test 2. In addition, (a) is a tissue-containing artificial dermis containing nine skins (magnification: 39 times), (b) is skin 2
Tissue-containing artificial dermis containing 5 cells (magnification 14 times), and (C) shows a bilayer artificial dermis without skin tissue for comparison.

[Explanation of symbols]

 Reference Signs List 1 hole 2 collagen sponge layer 3 silicone layer 4 skin tissue 5 epidermal tissue 6 dermal tissue

Claims (4)

[Claims] An artificial dermis for temporarily treating a full-thickness skin defect wound, wherein a silicone layer is provided on one side of a collagen sponge layer having a concave portion or a hole portion for accommodating skin tissue including epidermis and dermis tissue. Artificial dermis consisting of layers. 2. An artificial dermis in which a silicone layer is laminated on one surface of a collagen sponge layer having a concave portion or a pore portion, wherein an epidermal tissue and a dermal tissue are arranged in the concave portion or the pore portion in order from the silicone layer side. 2. A tissue-containing artificial dermis comprising a skin tissue including an epidermis and a dermis tissue. 3. A full-thickness skin defect wound of an artificial dermis in which a silicone layer is laminated on one side of a collagen sponge layer having a concave portion or a hole portion for accommodating skin tissue including epidermis and dermis tissue. Use for producing the tissue-containing artificial dermis according to claim 2. 4. The method according to claim 1, further comprising the step of placing skin tissue including epidermis and dermis tissue in a recess or a hole of the artificial dermis in which a silicone layer is laminated on one surface of a collagen sponge layer having a recess or a hole. Item 4. The method for producing a tissue-containing artificial dermis according to Item 2.