Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Solid support and method of culturing cells on said solid...
Reexamination Certificate
1999-07-16
2002-08-27
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Solid support and method of culturing cells on said solid...
C435S325000, C435S405000, C424S445000, C424S447000, C424S537000
Reexamination Certificate
active
06440740
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a material for activating adhesion, multiplication and cell-layer formation of epidermal cells of human and mammalian types.
The present active material is useful as an epidermal cell-culture bed and a defect skin repairing material or wound coating material for vital tissue.
BACKGROUND TECHNOLOGY
Cells are capable of obviously or latently self-multiplying as an essential unit of life, while each of organogenetic cells in multicellular organisms differentiates into various functional cells in a predetermined manner of multiplication.
In the case of the epidermis which protects an organism from the outside environment, cells are formed as a laminar layer to cover dermis situating under the epidermis and multiply only by adhering to other vital tissues or artificial materials to form a planar layer.
More specifically, the human epidermis comprises several layers and unevenly comes in contact with the dermis under the cellular layers through a basal layer so that the epidermis has a larger contact surface to maintain a close relationship with the dermis.
The epidermis has a so-called sebaceous membrane as a thin outermost lipid layer which functions to prevent water from evaporating, and under the membrane there are water-containing keratinous layers mainly consisting of keratin.
Each cell is arranged as a layer under the keratinous layer to form a cellular layer which comes in contact with a basal layer.
These cells repeatedly divide on the basal layer to develop new cells. That is, the basal layer is a base where new cells are formed and developed.
Epidermal cells thus formed in the basal layer gradually rise to the surface and then die when they arrive at the skin surface, thereby cell nuclei disappearing to form a keratinous layer.
As described above, the basal layer is located in the innermost part of the epidermis as a complicatedly uneven layer to form the contact surface so as to keep a close relationship with the epidermal cellular layer.
Although the formation and multiplication of epidermal cells on the basal layer was unsuccessful for reproduction in vitro for a long time, it has recently been possible to conduct the in vitro multiplication thereof because various kinds of solid surface base-beds for adhering epidermal cells were developed.
Even in such an in vitro multiplication, however, it has been quite difficult to form a plain cellular layer similar to that of in vivo multiplication for the lack of mutual adhesion of the cells.
Synthetic high polymer materials, such as polystyrene, natural high polymer materials such as collagen, and synthetic high polymer materials coated with natural materials have been conventionally developed as a cell culture bed. However, as these materials are not always the multiplication of epidermal cells, development of a novel cell culture bed has been desired.
Recently, there was proposed a cell culture bed comprising a water-insoluble silk membrane as a natural high polymer (see, JP-A No. 4-41,595).
The above mentioned cell culture bed is a &bgr;-type silk membrane prepared by crystallizing an amorphous silk membrane with an alcohol to water-insolubilize it, and which the animal cellular adhesion rate is improved compared with a conventional polystyrene culture bed.
As a matter of course, improved adherence to the bed is essentially necessary to epidermal cell cultures, while it is also required to form a plain cellular layer in which adjacent cells strongly adhere with each other.
Particularly, to apply such an epidermal cell culture technique to a wound healing method, it is still expected to develop a culture bed which promotes formation of a plain cellular layer.
Accordingly, it is an object of the present invention to provide a novel material for activating epidermal cell multiplication in which are effective adhesion and multiplication of human and mammalian epidermal cells to a base-bed surface obtained but also intercellular adhesion is promoted to form a plain cellular layer.
A further object of the present invention is to provide a novel culture bed in which in vitro formation of human and mammalian epidermal cellular layers can be conducted.
A still further object of the present invention is to provide a wound coating material for healing a skin defect region or wound of humans and mammals.
Another object of the present invention is to contribute to the basic study of epidermal functions by conducting the in vitro development of epidermal cells and also to the investigation of the action mechanism of skin defect healing by means of a novel material for activating epidermal cell multiplication.
DISCLOSURE OF THE INVENTION
The inventors have investigated in detail the interaction between crystal form of silk protein and epidermal cells and finally found that silk protein of a crystal form having a backbone helix structure, and especially &agr;-type or &agr;-helix silk fibroin is an outstanding material for achieving the above mentioned objects.
The present invention is based on this information and provides a material for activating epidermal cell multiplication, an epidermal cell culture bed in which the present material is used as a culture bed for cell multiplication, and a wound coating material in which the present material is used on the wound.
According to the present invention, there is provided (1) a material for activating epidermal cell multiplication which comprises silk fibroin of the &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent.
There is also provided (2) a material for activating epidermal cell multiplication which comprises silk fibroin of &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent and 50% by weight or less of a synthetic polyamino acid of an &agr;-helix crystalline form.
There is also provided (3) a material for activating epidermal cell multiplication described in item (1) or (2) in which said material is in the form of a fiber, membrane or powder.
There is also provided (4) a material for activating epidermal cell multiplication in which a solid surface of organic or inorganic substances is coated with a coating containing silk fibroin of &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent.
There is also provided (5) an epidermal cell culture bed which comprises a coating containing silk fibroin of &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent.
There is also provided (6) an epidermal cell culture bed in which a solid surface of organic or inorganic substances is coated with a stuff containing silk fibroin of &agr;-type or &agr;-helix X-ray diffraction pattern as a principal constituent.
There is also provided (7) an epidermal cell culture bed described in item (5) or (6) in which said bed is in the form of a membrane.
There is provided (8) a wound coating material which comprises a silk fibroin of &agr;-type or having &agr;-helix X-ray diffraction pattern as a principal constituent.
There is also provided (9) a wound coating material which comprises silk fibroin of &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent and 50% by weight or less of a synthetic polyamino acid of &agr;-helix crystalline form.
There is also provided (10) a wound coating material described in item (8) or (9) in which said material is in the form of a fiber, membrane or powder.
There is also provided (11) a wound coating material in which a solid surface of organic or inorganic substances is coated with a coating containing silk fibroin of &agr;-type or having an &agr;-helix X-ray diffraction pattern as a principal constituent.
A material for activating epidermal cell multiplication of the present invention is a material in which human and mammalian epidermal cells effectively adhere to a basal surface and multiply while forming a plain layer as a result of intercellular adhesion, i.e., a material in which adhesion and multiplication of epidermal cells as well as formation of a
Takasu Yoko
Tsubouchi Kozo
Yamada Hiromi
Flynn ,Thiel, Boutell & Tanis, P.C.
Japan as represented by Director General of National Institute o
Lankford , Jr. Leon B.
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