JP3094125B2 - Epidermal cell proliferation activation material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an activating material for promoting the attachment, proliferation and formation of a cell layer of human and mammalian epidermal cells. This activated material can be used as an epidermal cell culture bed, as a material for repairing defective skin in a living body, or as a wound dressing.

[0002]

2. Description of the Related Art Cells, which are basic units of life, have the ability to proliferate or potentially self-proliferate. In a multicellular organism, each cell constituting the living body has various functions. Differentiated, each showing a specific mode of proliferation. Of these, in the epidermis that protects the living body from changes in the external environment, cells are formed so as to cover the dermis below the epidermis in a layered manner, and only adhere to other living tissues or substances that substitute for them. It has the property of proliferating and forming a layer in a planar shape.

[0003] That is, in the case of the human epidermis, the epidermis is formed of several layers, and comes into close contact with the dermis under the cell layer through a basal layer in a wave (irregular) manner, thereby forming a close relationship. To maintain a large contact surface. The epidermis has a layer of oil, which has a thin water retention function called a sebaceous membrane on the outermost side. Under the sebaceous membrane, a stratum corneum (mainly composed of keratin) which can sufficiently contain water is formed. ing.

[0004] Under the stratum corneum, cells are arranged in layers to form a cell layer, which is then connected to the basal layer. At the interface with the basal layer, cells repeatedly divide to create new cells. In other words, the basal layer is where cells are born and generated. The epidermal cells generated in the basal layer are gradually pushed out to the surface side and eventually reach the skin surface, at which point the cells die and the cell nuclei disappear and become the stratum corneum.

[0005] The basal layer forms complex irregularities at the innermost part of the epidermis as described above, and forms a contact surface so as to maintain a close relationship with the epidermal cell layer. The generation and proliferation of epidermal cells initiated in the basal layer has been unsuccessful for a long time except in living tissue.However, as a result of exploring various solid surface basal floors for attaching epidermal cells, it has only recently become possible. Thus, it has become possible to grow epidermal cells in vitro.

[0006] However, even when the cells are grown by these methods, since the cells do not adhere to each other, it has been extremely difficult to form a planar cell layer conforming to the formation of an epidermal cell layer in a living body. Conventionally, as cell culture beds, synthetic polymer substances such as polystyrene, natural polymer substances such as collagen, and those obtained by corning synthetic polymer substances with natural polymer substances have been developed. It is not suitable for cell growth, and a new cell culture bed has been desired.

Recently, a cell culture bed made of a water-insoluble silk film has been proposed as a natural polymer (see Japanese Patent Publication No. 4-41595). This cell culture bed is a β-type silk film obtained by crystallizing a non-crystalline silk film with alcohol and insolubilizing the water, and the adhesion rate of animal cells is superior to that of a conventional polystyrene culture bed. Is done. However, in the culture of epidermal cells, although good adhesion is required, adjacent cells must strongly adhere to each other and form a layer in a planar shape. In particular, in applying the epidermal cell culture technology to wound treatment, there is a demand for the development of a culture bed that promotes the action of forming a planar cell layer.

[0008]

DISCLOSURE OF THE INVENTION The present invention is intended to provide not only a human and mammalian epidermal cell, which efficiently attaches and proliferates to the surface of a basal floor, but also promotes adhesion between cells to form a planar layer. It is an object of the present invention to provide a novel epidermal cell proliferation activating material that can be used.

[0009] Another object of the present invention is to provide a novel culture bed which enables the reproduction of epidermal cell layers of humans and mammals in vitro. Further, another object of the present invention is to provide a wound dressing for healing a skin defect (wound) in humans and mammals.

[0010] From another point of view, the present invention contributes to basic research on epidermal function by trying to reproduce epidermal cells in vitro, and uses a novel epidermal cell proliferation-activating material for skin. It is also intended to contribute to the study of the mechanism of action of healing of a defect.

[0011]

The present inventors have carefully studied the interaction between the crystal form of a silk protein and epidermal cells. As a result, the present inventors found that a silk protein having a helical structure in the main chain, particularly α-type silk protein. Alternatively, it has been found that α-helix silk fibroin is much superior in achieving the above object. The present invention has been achieved based on this finding, and it is intended to use an epidermal cell growth activating material, an epidermal cell culture bed using the same as a culture bed for cell proliferation, and a dressing material for a wound. To provide a wound dressing.

That is, the present invention resides in (1) a material for activating epidermal cell proliferation comprising a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix.

(2) The material containing silk fibroin as the main component, which shows an α-type or α-helix X-ray diffraction pattern, contains not more than 50% by weight of a synthetic polyamino acid exhibiting the α-helix crystal form. The epidermal cell proliferation activation material is made to exist.

Further, (3) the material for activating epidermal cell proliferation according to the above (1) or (2), which has a fibrous, film-like or powdery form.

(4) An epidermal cell comprising an organic or inorganic solid surface coated with a material containing, as a main component, silk fibroin whose X-ray diffraction pattern shows an α-type or α-helix crystal form. Exists in growth activating material.

(5) An epidermal cell culture bed composed of a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix.

(6) An epidermal cell comprising an organic or inorganic solid surface coated with a material containing, as a main component, silk fibroin whose X-ray diffraction pattern shows an α-type or α-helix crystal form. Present in the culture bed.

[0018] Also, (7) the epidermal cell culture bed according to (5) or (6), which has a film form.

(8) A wound dressing comprising a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix.

(9) In a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix, 50% by weight or less of a synthetic polyamino acid exhibiting a crystal form of α-helix. The wound dressing material contained.

The present invention also provides the wound dressing according to (8) or (9), which has (10) a fibrous, film-like or powdery form.

Also, (11) a wound dressing material obtained by coating a solid surface of an organic substance with a material containing as a main component silk fibroin whose X-ray diffraction pattern shows an α-type or α-helix crystal form. Exist.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The epidermal cell proliferation activating material of the present invention means that epidermal cells of humans and mammals efficiently adhere to and proliferate on the surface of a basal floor, and adhesion between cells forms a layer in a plane. A material that can be made and that promotes the attachment, proliferation and cell layer formation of epidermal cells. Therefore, the concept includes an epidermal cell culture bed and a wound dressing. The shape and structure are not particularly limited, and various shapes and structures such as a film (sheet or film), powder, bead, and fiber can be adopted.

The raw materials for producing the epidermal cell proliferation activating material include: a) a raw material selected from cocoon thread, raw silk, silk fabric, silk thread (fibroin fiber) or an unrefined product thereof; b) cocoon thread, raw silk Fibroin or sericin separately prepared from silk fabric, silk thread or unscrutinized products thereof; c) fibroin or sericin separately prepared or prepared from raw materials selected from cocoon thread, raw silk, silk fabric, silk thread or unscrutinized products thereof And d) a protein fiber substance spiked by silkworms such as silkworms and wild silkworms, such as used products such as fibers, powders, and films containing sericin and fibroin; Liquid silk etc. are all targeted. From the viewpoint of phase separation, the amount of sericin contained in these raw materials is preferably 40% or less, particularly preferably 20% or less.

In addition, from the viewpoint of moldability,
A synthetic polyamino acid capable of taking a helical structure (for example,
When the main chain repeating unit is -A-, -AG-, -AG-
S-, -AGGAS-, etc., wherein A is alanine, G is glycine, and S is sericin)
Mixing is performed within a range not exceeding 0% by weight. For example, if the raw material is fibroin separated and prepared,
To the extent not exceeding 50% by weight, a synthetic polyamino acid capable of forming the above helical structure is mixed. The material containing the silk fibroin of the present invention as a main component refers to a material containing silk fibroin obtained by converting silk fibroin in the above raw material into an α-type or α-helical structure by a gelatinization treatment method described below. .

[Method for pregelatinization] The above raw materials containing fibroin and sericin are scoured, then dissolved in an aqueous solvent in the presence of a neutral salt, and the aqueous solution of fibroin obtained by desalting is applied to a smooth solid surface. By spreading and drying, a water-soluble dried silk film is prepared. Next, in order to convert the water-soluble dried silk film into a water-insoluble silk film (sheet-like or film-like) having an α-type or α-helix structure in a crystalline form, the silk film has a water content of 40% at room temperature. Environmental humidity (for example, a relative humidity of 80 to 90%
) And left for a long time (for example, about 12 to 24 hours).

There are the following two methods. 1) Method of crystallizing in the drying process After casting the aqueous fibroin solution on a smooth solid surface, in the drying process, the water content of the aqueous fibroin solution is 40% ± 15.
When dried to a% state, this state is maintained for a long time. 2) Method of crystallizing after drying After fibroin aqueous solution is once dried, it is 40% ± at room temperature.
Crystallization is maintained in an environment of about 15% as it is for a long time.
The water-insoluble silk film having the α-type or α-helix structure thus manufactured can be dried and pulverized to obtain a powdery material.

Here, in the silk film, attention must be paid to the thickness of the silk film. When the thickness of the silk film is small, the molecular form of the silk film is affected by the material of the coating base, and the desired crystal form may not always be satisfied. On the other hand, if the thickness of the silk film is thick,
The film is easily peeled. From such a viewpoint, the thickness of the silk film is preferably 0.05 to 20 μm, and more preferably 0.5 to 10 μm.

[0029]

According to the present invention, there is provided a material having extremely excellent properties in terms of cell adhesion rate and proliferation, adhesion between cells, extensibility of a cell layer, and the like, which are basic requirements for activation of epidermal cells. can do. Therefore, it can be expected to greatly contribute to important problems in epidermis science, that is, research on biochemical reactions that occur during the formation of a cell layer of the epidermis, and application of epidermal cell culture technology to wound skin medicine.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it is to be understood that the present invention is not necessarily limited to Examples.

[0031]

EXAMPLES [Example 1] Raw silk of silkworm was boiled at a bath ratio of 100% with a 0.1% Na2CO3 aqueous solution for 2 hours, washed, dried and scoured. The silk thread (with a fibroin content of 97% or more) from which sericin was almost removed was dissolved in a solution having a molar ratio of calcium chloride, ethanol and water of 1: 2: 8, and the solution was dialyzed against water and desalted. Make an aqueous solution of silk fibroin. This aqueous solution is placed in a cell culture container (made of polystyrene; flat bottom, diameter 35 mm) and dried at room temperature to form a silk fibroin film (film shape) on the bottom of the container.

Then, the cell culture vessel having the above-mentioned silk fibroin film formed on the bottom is left at room temperature in an environment having a relative humidity of about 90% to adjust the water content of the silk fibroin film to about 40%. . Then, it is left as it is for 20 hours to perform the α-forming treatment.

Next, a part of the fibroin film obtained by the pre-gelatinization treatment was peeled off and taken out, and an X-ray diffraction diagram was taken. The result is shown in FIG. From this X-ray diffraction diagram, α
It can be understood that a silk fibroin film converted into a crystal structure of the α-type was obtained (for details of the determination of the crystal structure of the α-type, see “Polymer Papers” Vol. 33, No. 8).
(1976), pp. 453-462. )

Next, about 50,000 human epidermal cells were placed in the above-mentioned cell culture vessel on which the silk fibroin film converted to the α-type crystal structure was formed in a 1 ml culture solution [culture solution, medium for epidermal cells, The suspension was placed in Pharmaceutical Industry Co., Ltd. K110], and the suspension was statically cultured in a 37 ° C. incubator containing 5% of carbon dioxide gas saturated with steam and 95% of air.

FIG. 3 shows phase contrast micrographs (× 100) of the cells 5 hours after the start of the culture and 20 hours after the start of the culture. Also, after 20 hours of culture, the culture solution on the growing cells which adhered to the container was removed, the floating cells and dead cells were washed off with a physiological saline, and then a trypsin solution was added to the cells for 3 hours.
The epidermal cells adhered were kept at 7 ° C. to separate them. And
The number of epidermal cells attached was counted under a microscope, and the degree of epidermal cell adhesion [epidermal cell adhesion rate (%)] was examined. Table 1 shows the results.

[0036]

[Table 1]

Here, epidermal cell adhesion rate (%) = number of adhered cells after 20 hours / number of cells initially placed in cell culture vessel × 10
Indicated by 0. As an additional experiment, a similar static culture experiment was performed on a mixture containing 35% by weight of a synthetic polyamino acid exhibiting an α-helix crystal form in the above-mentioned silk fibroin film converted into the α-type crystal structure. Was done. The result was almost the same as the silk film (α type) shown in FIG. 3 and Table 1.

Example 2 In this example, for comparison, a silk fibroin β-type crystal powder was compared with a wound dressing. Α- and β-type crystal powders of silk fibroin were obtained as follows. First, the aqueous solution of silk fibroin obtained by the treatment of [Example 1] was cast on an acrylic plate and air-dried to form an amorphous film having a thickness of about 30 µm. And
It was crystallized into α-form and β-form as described below.

In the crystallization, pregelatinization was carried out by leaving the fibroin film in a room at room temperature and a relative temperature of about 90% for one day so that the water content in the fibroin film became about 40%. The β-formation was performed by spraying a 70% aqueous solution of ethyl alcohol on the fibroin film. These α-type and β-type fibroin films were dried and physically pulverized to obtain powders each having an average particle diameter of 50 μm or less.

An experiment was conducted on dogs (hairless dogs) to see the healing effect of using the α-type and β-type crystal powders of these silk fibroins as wound dressings. The back skin of the hairless dog was peeled off in three places to an area of 1 cm ^{2 to} the dermis. Each of three wound portions was disinfected by dropping a few drops of chlorhexidine glyconate 0.05% solution.

Next, one of the three wounds was coated with α-type crystal powder, and the other was coated with β-type crystal powder.
The remaining one site was kept in a sterilized state as a control. Thereafter, these three wounds were protected with a moisture-permeable film-like protective material (trade name, Tegaderm).
Table 2 shows the state of skin tissue regeneration at the wound portion after one week.

[0042]

[Table 2]

The healing effect is larger when the wound is covered with α-type crystal powder or β-type crystal powder than in the control group where no wound dressing was used. On the other hand, in the comparison between the α-type crystal powder and the β-type crystal powder, no clear difference is observed by visual observation,
Separately, close observation of the microstructure image revealed that the former α-type crystal powder showed a better healing process than the latter β-type crystal powder.

[0044]

[Comparative Example] [Comparative Example 1] (β-treatment) Instead of the α-treatment in Example 1, the β-treatment (the silk fibroin film formed on the bottom of the container before the α-treatment was dried, and then , A 70% aqueous solution of ethanol was sprayed and then dried at room temperature).
A crystallized silk fibroin film was prepared. The X-ray diffraction pattern is shown in FIG. FIG. 4 shows phase contrast micrographs (× 100) of the cells 5 hours and 20 hours after the start of the culture.
Here, as in Example 1, 20 minutes after the start of the culture.
The epidermal cell adhesion rate (%) at the time was examined. Table 1 shows the results.
Shown in

Comparative Example 2 (Polystyrene) Commercially available polystyrene container (specific description: Becton)
Dickinson Company FAL
CON1008) was directly used, and human epidermal cells were statically cultured in the same manner as in Example 1. In addition, a phase contrast micrograph (100 hours) of the cells 5 hours and 20 hours after the start of the culture.
5) is shown in FIG.

Here, in the same manner as in Example 1, the epidermal cell adhesion rate (%) was examined 20 hours after the start of the culture. Table 1 shows the results.

As shown in FIGS. 6 to 8 (schematic views of FIGS. 3 to 5 from above; α-type, β-type and polystyrene) and Table 1, the material for activating epidermal cell proliferation of the present invention is shown in FIG. When the human epidermal cells were cultured using the (culture bed), the attachment and proliferation of the cells to the culture bed surface were made more uniform, and the adhesion between the cells was improved, as compared with the cases of Comparative Examples 1 and 2. It can be seen that the growth extensibility of the cell layer is extremely excellent.

[0048] This is because the surface of the epidermal cell growth activating material having a helical structure forms a fine uneven surface similar to the membrane of the underlying layer of the living skin (the underlying membrane), and the surface of the material adheres to the surface of the material. It is considered that the coordination is tightly performed, and the adhesion and proliferation are performed uniformly and efficiently, and as a result, a cell layer similar to the cell layer of the living epidermis is formed. FIG. 6 (α
(Type), there is almost no gap between cells, and in FIG. 7 (β type), there is considerable gap between cells, and FIG. 8 (polystyrene)
In this case, a phenomenon occurs in which the contact between cells is small and the outer shape of the cells is not uniform.

[Brief description of the drawings]

FIG. 1 shows an X-ray diffraction photograph of α-form of silk fibroin of Example 1 in the present invention.

FIG. 2 shows an X-ray diffraction photograph of β-form of silk fibroin of Comparative Example 1 in the present invention.

FIG. 3 shows a phase contrast micrograph of the cells of Example 1 of the present invention.

FIG. 4 shows a phase contrast micrograph of the cells of Comparative Example 1 in the present invention.

FIG. 5 shows a phase contrast micrograph of the cells of Comparative Example 2 in the present invention.

FIG. 6 is a schematic diagram of the cells of Example 1 of the present invention.

FIG. 7 is a schematic diagram of a cell of Comparative Example 1 of the present invention.

FIG. 8 is a schematic diagram of a cell of Comparative Example 2 of the present invention.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12M 3/00 A61L 15/04 (73) Patent holder 597143731 Yoko Takasu 2--14-301-504 Namiki 2-chome, Tsukuba-shi, Ibaraki (72) Inventor Kozo Tsubouchi 6-15-8, Matsumaedai, Moriya-machi, Kita-Soma-gun, Ibaraki Pref. (72) Inventor Hiroo Yamada 1-8-10 Ninomiya, Tsukuba-shi, Ibaraki Pref. Ichinamiki 2-chome 14-301-504 (56) References JP-A-1-120283 (JP, A) JP-A-56-40156 (JP, A) JP-A-6-507304 (JP, A) Biopolymers, 1997 , Vol. 41, No. 2, p. 193-203 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5/06 A61K 38/17 A61L 15/64 C07K 14/435 C12M 3/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A material for activating epidermal cell proliferation comprising a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix. 2. A material containing, as a main component, silk fibroin showing an X-ray diffraction pattern of α-form or α-helix,
5 synthetic polyamino acids exhibiting the α-helix crystal form
An epidermal cell proliferation activating material containing 0% by weight or less. 3. The material for activating epidermal cell proliferation according to claim 1, which has a fibrous, film-like or powdery form. 4. An epidermal cell proliferation activating material comprising an organic or inorganic solid surface coated with a material containing, as a main component, silk fibroin whose X-ray diffraction pattern shows an α-type or α-helix crystal form. 5. An epidermal cell culture bed comprising a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix. 6. An epidermal cell culture bed in which an organic or inorganic solid surface is coated with a material containing silk fibroin as a main component, the X-ray diffraction diagram of which shows an α-type or α-helix crystal form. 7. The epidermal cell culture bed according to claim 5, which has a film form. 8. A wound dressing comprising a material containing silk fibroin as a main component, which shows an X-ray diffraction pattern of α-form or α-helix. 9. A material containing, as a main component, silk fibroin showing an X-ray diffraction pattern of α-form or α-helix,
5 synthetic polyamino acids exhibiting the α-helix crystal form
A wound dressing material containing 0% by weight or less. 10. The wound dressing according to claim 8, which has a fibrous, film-like or powdery form. 11. An X-ray diffraction pattern of the solid surface of an organic
A wound dressing material coated with a material containing silk fibroin as a main component, which exhibits a crystalline form of α-helix or α-helix.