JPH04129563A - Artificial skin and its manufacture - Google Patents

Abstract

PURPOSE:To decompose and absorb only a film with a body fluid such as an exudation in a cover for a burn and the like and regenerate a skin by heat- treating the collagen with a cross-link structure under the existence of water to obtain a film layer made of denatured collagen. CONSTITUTION:Collagen derived from cow corium is processed with acid or alkali, the collagen with a triple-bridge helix thus obtained is heat-treated or processed with a cross-linking agent, then it is heated at 50-125 deg.C under the existence of water to obtain a film made of denatured collagen. The telopeptide at molecule terminals is digestion-removed from the collagen processed with acid or alkali via prochtase or pepsin treatment to eliminate antigenicity for the raw material collagen. For the heat treatment, collagen is held for 24hr or longer at 110 deg.C in vacuum to be heat-dehydrated and cross-linked. When an artificial skin is to be generated, part of a patient to be grafted is peeled with a dermatome, it is divided into epidermis and corium, epidermis basal cells are put on the denatured collagen film thus obtained, fibrous bud cells are brought into contact with collagen sponge, and they are maintained under physiological conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an artificial skin used for treating skin wounds such as burns.

[Conventional technology]

BACKGROUND OF THE INVENTION In wounds in critical areas of human skin, especially in the case of severe burns, it is necessary to cover the wound with a substance that has some of the functions of skin. These features mean reduced fluid loss, infection prevention, and reduced potential scar area. The approach that has been taken to solve this problem is allogeneic transplantation,
Including the use of synthetic polymer structures or collagen films. Currently, the best treatment for the affected area is to take skin from a normal area of the patient and perform an autologous transplant. However, autologous transplantation is not always applicable; for example, when the defect is extensive, it becomes extremely difficult, and even when it is applicable, it is impossible to transplant to all the defects at once. Therefore, it is necessary to repeat transplantation many times over a long period of time. For this reason, there is a desire to develop artificial skin that can temporarily or permanently cover the affected area in place of autologous transplants, proliferate tissue cells, and promote tissue repair.

Furthermore, in recent years, a culture transplantation method or a method of transplanting a material similar to skin, which can be called cultured skin, has been carried out. This is aimed at severely ill patients, and the skin from the patient's remaining normal area is taken, and the isolated cells are cultured in a test tube, and the cells are multiplied tens to hundreds of times the size of the original cells. Later, it is transplanted together with a covering membrane to a patient's wound site to form epidermis and promote healing. As an example of clinical application of this method, Galico et al.
al., New England Journal of Medicine, Vol. 311, No. 7, 448-45.
1 page) (New Eng, J, Med, + Vol.
, 3+1. Na7P, 448-45m984)
).

[Problem to be solved by the invention]

Conventionally, collagen, which is a bio-derived material, has been molded into a sponge or film and used as a wound dressing.

However, when burns and the like are covered with these, there is a problem in that early tissue repair is not observed. In particular, a significant problem within these approaches is rejection of skin replacement, especially in the presence of immunosuppressants, and degradation of the graft by host enzymes.

On the other hand, a method of incorporating cells into collagen has been used, but the diameter of the collagen gel immediately after culturing is 60 mm, but it shrinks to about 10 mm after a week, so it is used as artificial skin. Difficult to apply to extensive burns.

When a cultured epidermal sheet is transplanted to a patient's wound site, it mainly survives when transplanted to the mother bed where the dermis remains, but it is difficult to engraft on the side of a full-thickness skin defect or on the wound surface of a third-degree burn. . Furthermore, when epidermal cells are cultured and transplanted onto an atelocollagen sheet, there is a problem that, because the collagen sheet has low substance permeability, sufficient nutrients are not supplied, and the epidermal cells eventually die.

[Means to solve the problem]

In view of the above circumstances, the present invention cultivates the epidermis and fibroblasts of a transplanted person in vitro to form skin tissue without shrinkage, and when covering burns, etc., only the film can absorb exudate and other body fluids. The purpose of the present invention is to provide an artificial skin base material that can be decomposed and absorbed by the skin to enable skin regeneration.

The above object of the present invention is achieved by the following configuration.

1) Artificial skin comprising a film layer of denatured collagen obtained by heat-treating collagen having a crosslinked structure in the presence of water.

2) Artificial skin consisting of a film layer of denatured collagen obtained by heat-treating collagen with a crosslinked structure in the presence of water, and an epidermal layer formed by layering skin-derived epidermal cells on one side of the film layer.

3) The reconstituted fibrotic collagen film is heat treated or treated with a crosslinking agent, and then it is incubated for 50 min in the presence of water.
A step of preparing a collagen film layer by heat treatment at ~125° C., bringing skin-derived epidermal cells into contact with one side of the collagen film layer obtained in this way, maintaining it under physiological conditions, and placing it on the film layer. 3. The method for producing artificial skin according to claim 2, comprising the step of proliferating epidermal cells and forming a multilayered epidermal layer on the film layer.

4) A collagen/denatured matrix sponge layer containing at least 5-fold FF196 denatured collagen, and a denatured collagen film layer laminated thereon obtained by heat-treating collagen having a crosslinked structure in the presence of water. Artificial skin.

5) In the artificial skin of item 4, skin-derived surface cells are layered on the upper surface of the denatured collagen film layer to form an epidermal layer, and living tissues such as skin-derived fibers and other cells are present within the sponge layer. Formed artificial skin.

6) Heat-treat or treat the reconstituted fibrillated collagen film with a cross-linking agent, which is then incubated for 50 min in the presence of water.
a step of preparing a collagen film layer by heat treatment at ~125°C; a step of pouring a mixed solution of fibrotic collagen and denatured collagen into a container, placing the collagen film layer thereon and freeze-drying the collagen film layer; Item 5, characterized in that it comprises the steps of bringing skin-derived epidermal cells into contact with the film layer and skin-derived fibrous cells with the sponge layer, and then maintaining them under physiological conditions. The method for manufacturing the artificial skin described.

The denatured collagen film of the present invention is produced by treating collagen derived from bovine dermis with an acid or alkali, and then heat-treating or treating the resulting double-stranded collagen with a crosslinking agent in the presence of water. Obtained by heating at 50-125°C.

The raw collagen is preferably acid- or alkali-treated collagen, which is further digested with protase or pepsin to remove the telopeptide at the molecular end, thereby eliminating antigenicity.

In the case of heat treatment, the collagen is heated to 110°C under vacuum.
It is desirable to hold the above condition for 24 hours or more to carry out thermal dehydration crosslinking. When processing with a crosslinking agent, there are no particular restrictions on the crosslinking agent, and aldehyde crosslinking agents such as glutaraldehyde,
Isocyanate crosslinking agents such as hexamethylene diisocyanate, carbodiimide crosslinking agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the like are used.

If the collagen to be cross-linked is a dispersed water-soluble type with socks in the double strands, the physical properties will not improve much even if cross-linked, so the dispersed collagen should be prepared at 37°C using a phosphate buffer. It is preferable to perform a neutralization treatment to form reconstituted fibrotic collagen having a periodic fibrous structure similar to that found in vivo.

The cross-linked collagen is further heated to 50-50% in the presence of water.
It is heated at 125°C, preferably 90-121'C for 20 minutes to 1 hour. Films treated under these conditions do not degrade when epidermal cells are cultured, but when transplanted, they are quickly degraded and absorbed by exudates and other body fluids.

The collagen/denatured collagen matrix can be obtained by mixing the fibrotic collagen solution prepared above and the denatured collagen solution and freeze-drying the mixture. At this time,
The mixed solution is poured into a styrene container, the above-mentioned denatured collagen film is placed thereon, and the denatured collagen film is lyophilized to obtain a collagen/denatured collagen matrix laminated with the film.

To create artificial skin, first, the part of the patient to be transplanted is exfoliated with a dermatome, separated into the epidermis and dermis, and the epidermal basal cells are placed on the denatured collagen film obtained above.
Artificial skin can be obtained by bringing fibers and other cells into contact with the collagen sponge and maintaining it under physiological conditions.

This series of operations can be performed by experts who have experience in handling cells using only routine experiments.

〔Example〕

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

Example 1 <Preparation of denatured collagen film> 1.0 g of atelocollagen is dissolved in dilute hydrochloric acid of pH 3.0 to prepare a 0.3 (w/v)% solution. This solution was placed in a constant temperature bath at 4°C, and while stirring, phosphate buffer was added until the final concentration was 0. l(v/V)% atelocollagen, 30mM 2-storium phosphate, lOhMNaCj
A collagen solution No. 7 was prepared. Next, it was immersed in a constant temperature bath at 37° C. for one day to obtain a fibrotic atelocollagen solution. This solution was centrifuged at M (5000r, p, a, 10
minutes) and concentrated to prepare a 0 J (v/v)% fibrotic atelocollagen solution. This solution was poured into a styrene container and air-dried in a clean bench. This film was treated under vacuum at 140° C. for 24 hours to perform thermal dehydration crosslinking. This film was immersed in distilled water and heated in a sterilized bottle at 121"C for 30 minutes to remove the denatured collagen film.

Example 2 A crosslinked film prepared in the same manner as in Example 1 was immersed in distilled water and heat-treated at 50°C for 30 minutes to obtain a denatured collagen film.

Example 3 A crosslinked film prepared in the same manner as in Example] was immersed in distilled water and heat-treated at 70°C for 30 minutes to obtain a denatured collagen film.

Example 4 A crosslinked film prepared in the same manner as in Example 1 was immersed in distilled water and heat-treated at 90° C. for 30 minutes to obtain a denatured collagen film.

Comparative Example 1 A film was prepared in the same manner as in Example 1 to introduce crosslinking to obtain a film before heat treatment.

Test Example 1 <Characteristics of Various Collagen Films> The shrinkage rates of the films obtained in Examples 1 to 4 and Comparative Example 1 were calculated from the wet strength, helix content, and area ratio before and after heat treatment. The results are shown in Table 1.

The higher the surface hot water treatment temperature, the more the helical structure was destroyed and denatured.

Test Example 2 Degradation of collagen films by enzymes Each of the films obtained above was degraded at 37°C in a solution of 4 units/ml protease or +00 units/ml bacterial collagenase. The amount of degraded collagen was determined by acid hydrolyzing the eluted collagen degradation product and quantifying the hydroxyproline content by amino acid analysis. The results are shown in Figures 1 and 2.

The figure shows that the enzyme degradability of the collagen film varies depending on the hot water treatment conditions.

Example 5 <Formation of living tissue> The denatured collagen film prepared above was
The film was cut into a size of 2 cn+ and placed in a 60 mm petri dish. Next, epidermal cells collected from rat skin were incubated at a concentration of 1 x 107eel Is/ml (1
1 ml of the suspension (suspended in DME medium containing 0% tallow serum) was collected and further cultured at 37°C for 3 days.

A 2 cm x 2 cm defective wound was created on the back of the rat from which the cells were collected above, and the living tissue obtained above was transplanted thereto. It was confirmed that the collagen film of this living tissue was decomposed within one week after transplantation, and the epidermis came into direct contact with the mother's body, indicating that the tissue had survived.

Example 6 <Preparation of denatured collagen sponge laminated with denatured collagen> 0.3 (v/v)% atelocollagen solution and phosphate buffer (45mM NaH
Mix PO4150sMNacff) at a ratio of 1:2,
This was incubated at 37°C for 4 hours. Next, this solution was centrifuged at 500 Or, p, a for 10 minutes, and a precipitate was prepared to a concentration of 1 (v/v)%, and this was mixed with the denatured collagen prepared above. Furthermore, the mixed solution of fibrotic collagen and denatured collagen was poured into a styrene container, and the denatured collagen film prepared above was placed thereon, frozen at -30°C, and freeze-dried to obtain artificial skin.

Example 7 Formation of living tissue> Denatured collagen film/collagen produced above
The denatured collagen sponge was cut into a size of 2 cm x 2 cm and placed in a 60 mm petri dish with the denatured collagen film facing downward.

Next, fibrous cells lXl06ee collected from rat skin
The solution was prepared at a concentration of 1 Is/ml (suspended in DME medium containing 10% tallow serum), and 1 ml was plated on a denatured collagen film. Furthermore, 3 ml of culture solution was injected around the sponge and cultured at 37°C for 3 days. Next, turn this sponge over so that the denatured collagen film is on top, and place the epidermal cells collected from rat skin on top.]
1 ml was collected at a concentration of 107 cells/ml (suspended in DME medium containing 10% tallow serum), and
The desired artificial skin was obtained by culturing at 7°C for 311 hours.

A 2 cm x 2 cm defective wound was created on the back of the rat from which the cells were collected above, and the living tissue obtained above was transplanted thereto. This living tissue took root immediately after transplantation, and histopathological images on the 7th day of transplantation showed that something similar to skin tissue was being constructed.

〔Effect of the invention〕

This development is possible by incorporating epidermal cells, which are skin structural cells, into a film made of denatured collagen that has been heat-treated in the presence of water (1 mm) and maintaining them under physiological conditions. It makes it possible to form living skin tissue outside the body.When this living tissue is transplanted into a skin defect, the healing process can be significantly shortened.

Furthermore, the present invention incorporates epidermal cells and fibrous cells, which are skin constituent cells, into a matrix that combines a denatured collagen film and a denatured collagen/collagen sponge layer, and maintains them under physiological conditions to survive in vitro. This makes it possible to form a skin tissue that has a unique structure. When this living tissue is transplanted into the skin defect, some tissue has been constructed, so the healing process can be significantly shortened.

[Brief explanation of the drawing]

Figure 1 shows the amount of collagen dissolved after 30 minutes at 37°C after immersing a collagen film treated with warm water at various temperatures (50, 70, 90, 121"c) in a protease solution. Figure 2 shows the amount of collagen dissolved at 37°C for 30 minutes. Temperature (50, 70, 90°121°C
) The collagen film treated with warm water was immersed in a collagenase solution, and the amount of collagen dissolved after 24 hours at 37°C is shown. Figure Untreated 50°C 70'C 90'C +21'C Figure

Claims (1)

[Scope of Claims] 1) An artificial skin comprising a film layer of denatured collagen obtained by heat-treating collagen having a crosslinked structure in the presence of water. 2) Artificial skin consisting of a film layer of denatured collagen obtained by heat-treating collagen with a crosslinked structure in the presence of water, and an epidermal layer formed by layering skin-derived epidermal cells on one side of the film layer. 3) The reconstituted fibrotic collagen film is heat treated or treated with a crosslinking agent, and then it is incubated for 50 min in the presence of water.
A step of preparing a collagen film layer by heat treatment at ~125° C., bringing skin-derived epidermal cells into contact with one side of the collagen film layer obtained in this way, maintaining it under physiological conditions, and placing it on the film layer. 3. The method for producing artificial skin according to claim 2, comprising the step of proliferating epidermal cells to form a multilayered epidermal layer on the film layer. 4) A collagen/denatured matrix sponge layer containing at least 5% by weight of denatured collagen, and a denatured collagen film layer laminated thereon obtained by heat-treating collagen having a crosslinked structure in the presence of water. Artificial skin. 5) In the artificial skin according to claim 4, skin-derived surface cells are layered on the upper surface of the denatured collagen film layer to form an epidermal layer, and living tissue of skin-derived fibroblasts is present in the sponge layer. Formed artificial skin. 6) The reconstituted fibrillated collagen film is heat treated or treated with a crosslinking agent, and then it is incubated for 50 min in the presence of water.
A step of preparing a collagen film layer by heat treatment at ~125° C., a step of pouring a mixed solution of fibrotic collagen and denatured collagen into a container, placing the collagen film layer thereon and freeze-drying it, and a step of freeze-drying the collagen film layer. 6. The method according to claim 5, comprising the steps of bringing skin-derived epidermal cells into contact with the film layer and skin-derived fibroblasts with the sponge layer, and then maintaining them under physiological conditions. The method for manufacturing the artificial skin described.