JPH04129562A - Artificial skin - Google Patents

Abstract

PURPOSE:To decompose and absorb only a film with a body fluid such as an exudation and regenerate a skin by forming the film with a coacervate made of denatured collagen and water-soluble polysaccharide, and cross-linking the film. CONSTITUTION:Collagen derived from cow corium is digestion-processed with enzyme such as prochtase or pepsin, antigenic group telopeptide is removed to obtain atherocollagen, and it is heated and denatured at the temperature of 37-90 deg.C to obtain denatured collagen. Water-soluble chondroitin sulfate is particularly preferable among polysaccharide for the water-soluble polysaccharide. A coacervate made of denatured collagen and water-soluble polysaccharide is obtained by mixing a denatured collagen aqueous solution and a water-soluble polysaccharide aqueous solution at the ratio of denatured collagen 1 pts.wt. and water-soluble polysaccharide 0.05-1 pts.wt., adjusting pH to acidic with hydrochloric acid and the like, and air-drying the cloudy solution in a clean bench. It is heat-dehydrated and cross-linked for 24hr or longer within the range of l40-160 deg.C in vacuum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel artificial skin used for healing 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, Gallico et al.
New England Journal of Medicine, Vol. 311, No. 7, 448-45.
1 page) (New Eng, J, Med,, VoI
z, 311. No, 7. P, 448-451
(1984)).

[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 culture is 60 m1.
However, it shrinks to about IO after - weeks, making it difficult to apply to a wide range of burns when used as artificial skin.

When transplanting a cultured epidermal sheet to a patient's wound site, it is most likely to be transplanted to a wound site where the dermis remains, but it is difficult to transplant it to a wound with full-thickness skin loss or third-degree burns. 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.

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

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

1) Artificial skin consisting of a film formed from coacervate made of denatured collagen and water-soluble polysaccharide, and characterized in that the film is crosslinked.

2) Artificial skin in which a living tissue of skin-derived epidermal cells is formed on one side of the artificial skin of item 1.

3) An artificial skin comprising a sponge layer of a matrix comprising collagen and denatured collagen containing at least 5% by weight of denatured collagen, and the artificial skin of item 1 laminated thereon.

4) An artificial skin according to item 3, in which a living tissue of skin-derived epidermal cells is formed on the film made of the coacervate, and a living tissue of skin-derived fibroblasts is formed on the sponge layer. Skin.

The denatured collagen in the present invention is obtained by digesting collagen derived from bovine dermis with enzymes such as proctase or pepsin to remove antigenic telopeptides to obtain atelocollagen, which is then heated to a temperature of 37 to 90°C to denature it. Something is desirable.

This thermal denaturation condition greatly influences coacervate formation with polysaccharides. The degree of unwinding of the helical fibers (helices) of collagen molecules, the reactivity to pH changes, etc. differ depending on the feeding temperature and the degree of cystic treatment time. For this purpose, the degree of denaturation can be adjusted so that a coacervate is appropriately formed when mixed with a dilute aqueous solution of a water-soluble polysaccharide. Collagen has a thermal denaturation temperature of 37°C, and when a temperature of 37°C or higher is applied, the three-channel helical structure unique to collagen is lost and the structure changes. Complete randomization of the three-dimensional structure without chain scission also appears to be an important factor. A suitable heat denaturation treatment is 3 at 60°C.
This is a 0 minute treatment, and when this treatment is performed, the helix content of the denatured collagen is about 40%.

Water-soluble polysaccharides refer to polysaccharides that dissolve well in water, and mucopolysaccharides, particularly acidic mucopolysaccharides such as chondroitin sulfate, heparan sulfate, dermatan sulfate, hyaluronic acid, and heparin, are preferred, and alginic acid is also preferred. Preferably used.

Coacervate consisting of denatured collagen and water-soluble polysaccharide is produced according to a conventional method. That is, a denatured collagen aqueous solution and a water-soluble polysaccharide aqueous solution are mixed at a ratio of 0.05 to 1 part by weight of the water-soluble polysaccharide per 1 part by weight of the denatured collagen, and the pH is adjusted to acidic using hydrochloric acid or the like. Obtained by air-drying a cloudy solution in a clean bench.

In order to prevent the production of artificial skin from being degraded by enzymes such as collagenase, the film made of coacervate is heated under vacuum at a temperature in the range of 110 to 180°C, preferably 1.
It is preferable to carry out thermal dehydration crosslinking within the range of 40 to 160°C for 24 hours or more. Films treated under these conditions do not decompose while culturing epidermal cells, but when transplanted, they are quickly decomposed and absorbed by exudate and other body fluids.

In addition, coacervate film is used when culturing epidermal cells.
In consideration of the supply of nutrients from the culture medium, it is preferable to make it porous and provide air permeability. The film made of coacervate contains glucose (M-180) and vitamin B.
12 (M-H55), inulin CM = 5200)
can pass through solutes such as

The thickness of the coacervate film can be adjusted by adjusting the concentration and volume of the collagen solution. The film thickness is preferably about 5 to 100 ρ in relation to solute permeability.
More preferably, it is about 10 to 50 t1m.

Collagen/denatured collagen matrix can be obtained by mixing the fibrotic collagen solution prepared above and 8 liquids of denatured collagen, 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 remove the part of the patient to be transplanted using a dermatome, separate it into the epidermis and dermis, then bring the epidermal basal cells into contact with the coacervate film obtained above, and add fibroblasts to the collagen sponge. contact the cells,
Artificial skin is obtained by maintaining it under physiological conditions.

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

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

[Example] <Preparation of coacervate> Atelocollagen was swollen with distilled water to a concentration of 0.3 (v/v)% to obtain an atelocollagen solution. After this aqueous solution was allowed to warm to room temperature, it was kept at 60°C. A heating operation is performed in an oven for about 2 hours to obtain denatured collagen. This denatured collagen is kept above the denaturation temperature (37°C). Note that the denatured collagen obtained at this treatment temperature and time is within the range in which the helical structure can be regenerated at a certain rate after cooling. The helix content of the denatured collagen thus obtained was about 40%. On the other hand, chondroitin-6-sulfuric acid is dissolved in distilled water to give a chondroitin-6-sulfuric acid aqueous solution to a concentration of 1 (v/V)%. In addition, what is obtained as chondroitin-6-sulfuric acid is dissolved in water, and then the Na portion is replaced with an H+ type using an ion exchange resin. When mixing, the amount of chondroitin-6-sulfate added to the total amount of substances should be 5 (v/v)% or more.
v/v)% or less, preferably 10(v/w)%
30 (v/v)% or less.

Maximum yield of coacervate can be expected within this range.

After mixing, adjust pl+ using 1N HCj).

As the pH decreases, cloudiness occurs and coacervate droplets are formed. This solution was air-dried to dryness in a clean bench.

The film obtained above was evacuated under vacuum for 1 hour, the temperature was further raised to 140°C, and the vacuum was kept for 24 hours, after which the temperature was lowered and the sample was taken out.

<Permeability test of coacervate film> The solute permeability of the coacervate film obtained above was evaluated. Glucose (molecular weight + 180),
The permeability of vitamin B12 (molecular weight: 1355) and inulin (molecular weight: 5200) was investigated. 1st to 3rd
The figure shows the results of permeability tests for various solutes.

<Preparation of collagen/denatured collagen sponge laminated with coacervate> o, a (v/v)% atelocollagen solution and phosphate buffer (45+aM Na2HPO4, +50a+M Na
Cl1) were mixed at a ratio of 1=2, and this was incubated at 37°C for 4 hours. Next, add this solution to 5000r, p.
After centrifugation for 10 minutes at a+, the precipitate was reduced to 1 (w/v)%
This was mixed with the denatured collagen prepared above. Furthermore, pour the fibrotic collagen/denatured collagen mixed solution into a styrene container, place the coacervate film prepared above on top of it, and -
It was frozen at 30°C and freeze-dried.

Formation of a living tissue〉 (+) The coacervate film prepared above was
It was cut into a size of e+n x 2 cm, and a coacervate film was placed on a 60-inch shear plate. Next, epidermal cells collected from rat skin]
(suspended in DME medium containing 10% tallow serum) and cultured at 37°C for 3 days.

A defective wound measuring 2 cm x 2c+n was created on the back of the rat from which the cells were collected above, and the living tissue obtained above was transplanted thereto. In this living tissue, the coacervate film decomposes within one week after transplantation, and the epidermis comes into direct contact with the mother's body.
It was confirmed that the tumor had survived.

(2) The coacervate film/collagen-denatured collagen sponge prepared above was cut into a size of 2CI11 x 2 cm, and placed on a (10+U shear plate with the coacervate film facing down).

Next, fibroblasts lXl06ce collected from rat skin
lls/ml (suspended in DME medium containing 10% tallow serum) and 1 ml onto the coacervate.
Sowed. 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 coacervate film is facing up, and place the epidermal cells collected from rat skin on top of it.
Collect 1 ml of eel Is/ml (suspended in DME medium containing 10% tallow serum) and further incubate at 37°C for 3
Cultured for 1 day.

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〕

The present invention incorporates epidermal cells, which are skin constituent cells, into a film in which a mixture of denatured collagen and water-soluble polysaccharide forms a coacervate structure, and maintains them under physiological conditions to make them viable in vitro. It makes it possible to form skin tissue. Transplanting this living tissue into skin defects can significantly speed up the healing process.

Furthermore, the present invention incorporates epidermal cells and fibroblasts, which are skin constituent cells, into a matrix that combines a coacervate 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, a certain amount of tissue structure has been achieved, so the healing process can be significantly shortened.

[Brief explanation of the drawing]

FIG. 1 shows the glucose permeability of coacervate films. Figure 2 shows the vitamin B1□ permeability of coacervate films. Figure 3 shows the inulin permeability of coacervate films. In FIGS. 1 to 3, the thickness of the coacervate is 20B for O and 30B for Δ. IZll, mouth indicates 5o-. Time (hr) Fig. Time (hr) Fig.

Claims (1)

[Scope of Claims] 1) An artificial skin comprising a film formed from a coacervate made of denatured collagen and a water-soluble polysaccharide, the film being crosslinked. 2) An artificial skin in which a living tissue of skin-derived epidermal cells is formed on one side of the artificial skin of claim 1. 3) An artificial skin comprising a matrix sponge layer comprising collagen and denatured collagen containing at least 5% by weight of denatured collagen, and the artificial skin of claim 1 laminated thereon. 4) Claim 3 (7) Forming a living tissue of skin-derived epidermal cells on the film made of the coacervate of the artificial skin, and forming a living tissue of skin-derived fibroblasts on the sponge layer. Tightened artificial skin.