ARTIFICIAL SKIN AND ITS PRODUCTION

Abstract: An artificial skin comprising an insoluble atelocollagen sheet and an epidermal cell layer cultured only on one surface of the sheet, and a method of producing the same are disclosed, which method includes the steps of: forming an insoluble atelocollagen sheet; inoculating epidermal cells only on one surface of the atelocollagen sheet; and suspend­ ing the atelocollagen sheet in a liquid culture medium to culture the epidermal cells, thereby forming an epidermal cell layer.

Description: The present invention relates to an artificial skin, for transplanting onto a wounded skin surface such as a burned or scratched skin surface, and to its production. Conventionally, artificial skins made of collagen and so on are used in practice as temporary wound-covering materials. Cell-incorporating artificial skins having biocompatibility are proposed by E. Bell (Science 211 , 1052 (1981)) and I.V. Yannas et al. (Science 215 , 174 (1982)). However, it takes a long cell culture period (about one month) to obtain an artificial skin by this method and the size of artificial skin piece obtainable is limited since the agar-like collagen serving as the cell carrier is soft; accordingly these artificial skins are not used clinically. Currently, a method for fast culturing cells is proposed by Gallico G.G. et al. (NEJM 311 , 448-451 (1984)). According to this method, epidermal cells isolated from a skin piece are cultured using 3T3 (an established cell strain obtained from a Swiss mouse subcutaneous tissue) as supporting cells, and the cultured epidermal sheet is treated with dispase and removed from the supporting cells. The sheet is applied onto an ointmentcoated gauze for transplantation. In this case, although the cell culture period is shortened, the supporting cells remaining in the epidermal sheet adversely affect the recipient's body. This method also has the following defects: (1) The epidermal sheet must be removed by dispase treatment. (2) The area of the epidermal sheet is reduced to about ½ the original area upon removal. (3) A carrier, such as a gauze, for the epidermal sheet is additionally required. (4) Since only the epidermal sheet is transplanted, without a dermis component, scarring remains after healing. In the case of a critical disorder such as a deficiency in the deep dermal portion, sufficient effect cannot be expected by this method. According to the present invention an artificial skin comprises an insoluble atelocollagen sheet and an epidermal cell layer cultured on one surface of the sheet; and a method of producing an artificial skin comprises the steps of: forming an insoluble atelocollagen sheet; inoculating epidermal cells only on one surface of the atelocollagen sheet; and curing the epidermal cells on the sheet (e.g. in a liquid culture medium) to form an epidermal cell layer. The insoluble atelocollagen sheet used in the present invention is obtainable by air-drying an atelocollagen solution and treating the resulting sheet by molecular crosslinking such as ultraviolet irradiation or glutaraldehyde treatment. The term "atelocollagen" used herein means a collagen which is obtained by treating collagen with protease, such as pepsin, in an aqueous acid solution, such as aqueous acetic acid, to remove telopeptides at the terminal ends of collagen molecules and to cleave the molecular crosslinks. The artificial skin according to the present invention may comprise an insoluble atelocollagen sheet having a fibroblast layer cultured on at least one surface thereof and an epidermal cell layer on the fibroblast layer. In this case, fibroblasts obtained from the recipient's living body are preferably used. The artificial skin according to the present invention may comprise an insoluble atelocollagen sheet having an epidermal cell layer on one surface thereof and a fibroblast layer cultured on the other surface. The artificial skin of the present invention can be prepared by the following method: An atelocollagen solution is poured into an appropriate container, air-dried, and subjected to ultraviolet irradiation to form an insoluble atelocollagen sheet. Epidermal cells are inoculated only on one surface of the sheet. Alternatively, fibroblasts are cultured on at least one surface of the sheet to form a fibroblast layer, and epidermal cells are inoculated on the fibroblast layer. Subsequently, the resultant sheet is suspended in a liquid culture medium and the epidermal cells are cultured to form an epidermal cell layer, thereby producing an artificial skin of the present invention. The atelocollagen sheet used in the method of the present invention preferably has a high permeability in order to shorten the culture period for the epidermal cells. Such an atelocollagen sheet is obtainable in the following manner: An atelocollagen solution is poured into an appropriate container and air-dried to obtain an atelocollagen sheet, and the sheet is immediately allowed to be swollen with distilled water, subjected to ultraviolet irradiation for 10 to 30 minutes, and air-dried. Alternatively, the same atelocollagen solution as above is air-dried to obtain an atelocollagen sheet, and the sheet is subjected to ultraviolet irradiation, swollen with distilled water, and freeze-dried. The artificial skin according to the present invention has a two- to four-layer structure similar to that of a living body. It comprises an epidermal cell layer, an atelocollagen sheet, and preferably a fibroblast layer. The epidermal cell layer corresponds to the epidermal layer of the living body, and the atelocollagen sheet or the combination of the atelocollagen sheet and the fibroblast layer corresponds to the dermis layer of the living body. In the method of the present invention, when fibroblasts are cultured on at least one surface of the atelocollagen sheet to form a fibroblast layer and thereafter epidermal cells are inoculated thereon, the culturing and multiplying of the epidermal cells is accelerated because of the adhesion effect of the fibroblast layer and the atelocollagen sheet and the epidermal cell supporting effect of the fibroblast layer. It is preferable to irradiate the fivroblasts with X-rays before inoculating epidermal cells, so as to control proliferation of the fibroblasts. The artificial skin according to the present invention can have the following advantages: (a) The supporting cells do not adversely affect the recipient's body because the fibroblasts of the recipient's body are used as the supporting cells, (b) There is no need for enzymatic treatment such as dispase treatment, with consequent loss of artifical skin area. (c) No additional supporting body is required for the epidermal sheet. (d) When a fibroblast layer as a dermis component is incorporated, sufficient healing effect can be obtained even for a wound with a deficiency in the deep dermis portion, and no scarring remains after healing. The present invention will be more clearly understood by reference to the following examples; however, the examples are intended to illustrate the invention and are not to be construed to limit the scope of the invention. Example 1 An acidic solution containing 0.3 to 1.0% of a bovine dermis atelocollagen prepared in a germ-free manner was poured into a polystyrene mold frame (9 × 18 × 1 cm) such that the solution level was 1 to 5 mm, and was air-dried to form an atelocollagen sheet. The sheet was removed from the mold frame, and ultraviolet irradiation was applied to the two surfaces of the sheet to crosslink the atelocollagen molecules of the sheet, i.e., to render the sheet insoluble. This sheet was cut to give a circular sheet with a diameter of 25 mm. About 1 × 10⁵ epidermal basal cells isolated from the skin of a newborn rat were inoculated only on one surface of the circular sheet. The circular sheet was suspended in a liquid culture medium ("medium 199") containing a 10% fetal bovine serum, streptomycin, and a phosphoric acid buffer, and was allowed to stand in an incubator at a CO₂ concentration of 5% and a temperature of 37°C to culture the epidermal basal cells. In one week the epidermal cells covered one entire surface of the sheet. An artificial skin comprising two to three epidermal cell layers was thus obtained. The artificial skin obtained was transplanted onto a skin deficiency portion of a rat of the same type as the donor rat described above. After about three weeks the skin deficiency portion exhibited ideal healing with no skin contraction. Example 2 About 2 × 10⁵ fibroblasts obtained from the newborn rat of Example 1 were cultured on an atelocollagen sheet as in Example 1 to form a fibroblast layer. Subsequently, by same procedures as in Example 1, epidermal cells were cultured on the other surface of the circular sheet. The liquid culture medium used in this example consists mainly of a mixture of an Eagle minimum essential medium (modified by Dulbecco) containing a 10% fetal bovine serum and an antibiotic, and HAM F-12 at a ratio of 3: 1. The epidermal cells in this example multiplied faster than in Example 1 and covered one entire surface of the sheet in about 5 days. The artificial skin obtained in this manner was transplanted onto a skin deficiency portion of a rat of the same type as the donor rat. After about three weeks the skin deficiency portion exhibited ideal healing with no skin contraction. Example 3 By the same procedures aa in Example 2, but modified as follows, an atelocollagen sheet having good permeability was obtained. More specifically, an atelocollagen sheet obtained by air-drying an atelocollagen solution was subjected to ultraviolet irradiation for 10 to 30 minutes, swollen with distilled water, and freeze-dried. The atelocollagen sheet obtained in this manner was used to culture epidermal cells by the same procedures as in Example 2. The epidermal cells multiplied faster than in Example 2. The artificial skin of this example was thus obtained in four days. Example 4 About 2 × 10⁵ fibroblasts obtained from a newborn rat were cultured on one surface of an atelocollagen sheet prepared by the same procedures as in Example 1. About 2 x 10⁵ fibroblasts of the same type as above but irradiated with 5,000-rad X rays were separately cultured on the other surface of the atelocollagen sheet to form a fibroblast layer. Epidermal cells were cultured on the fibroblast layer under the same conditions as in Example 2. The epidermal cells of this example multiplied faster than Example 2 and covered one entire surface of the sheet in three days. The artificial skin obtained in this manner was transplanted onto a skin deficiency portion of a rat of the same type as the donor rat. The skin deficiency portion exhibited ideal healing in about two weeks with no skin contraction.

Claims: 1. An artifical skin comprising an insoluble atelocollagen sheet and an epidermal cell layer cultured on one surface of said sheet. 2. An artificial skin according to claim 1 wherein said atelocollagen sheet has a fibroblast layer cultured on at least one surface thereof and an epidermal cell layer on said fibroblast layer. 3. An artificial skin according to claim 1 wherein said atelocollagen sheet has a fibroblast layer cultured on the opposite surface thereof to that having said epidermal cell layer. 4. A method of producing an artificial skin comprising the steps of: forming an insoluble atelocollagen sheet, inoculating epidermal cells on one surface of said atelocollagen sheet; and culturing the epidermal cells on the sheet to form an epidermal cell layer. 5. A method according to claim 4, wherein fibroblasts are cultured on at least one surface of said atelocollagen sheet to form a fibroblast layer, and thereafter the epidermal cells are inoculated.

Inventors: KATSUTOSHI, YOSHIZATO, TERUO, MIYATA, TOSHIO TAIRA
Applicants: KOKEN CO. LTD.
Application year: 1987
Main classification: A61F2
IPC codes: A61F2, A61L27