WO2007001016A1 - Dermal tissue improving material and use thereof - Google Patents

Abstract

A material for improving a dermal tissue which mainly comprises a fibroblast derived from an oral tissue of human or other mammal. Preferably, the fibroblast is a human gingival fibroblast and/or a human periodontal fibroblast, which is excellent in capability of producing a vascular endothelial growth factor (VEGF) and/or a keratinocyte growth factor (KGF).

Description

 Specification

 Skin tissue improving material and use thereof

 Technical field

 [0001] The present invention relates to the use of cells derived from oral tissues, and more specifically to a skin tissue improving material (composition for improving skin tissue) mainly composed of the cells. The present invention also relates to a method for improving skin tissue using cells derived from oral tissues.

 Note that this international application claims priority based on Japanese Patent Application No. 2005-19 0374 filed on June 29, 2005, the entire contents of which are incorporated herein by reference. Is incorporated.

 Background art

 [0002] Various biomaterials are used as a skin tissue repair material to repair abnormalities or defects in skin tissue due to wounds or the like, or congenital or acquired (aged) wrinkles or depressions. ing. For example, in International Publication No. WO1997Z004720 (corresponding to JP 11-510069 A), dermal fibroblasts isolated from the patient's own skin tissue are cultured outside the body, and the culture is then applied to the skin tissue (affected area). The method of injecting is described. In addition, in the document of DeLustro F et al. (J Biomed Mater Res., 1986, No. 20 (1), p. 109-120), the C-terminal and N-terminal peptide parts are removed and the antigen is removed. It describes a method of injecting urushi collagen (atelocollagen) with reduced sex into the affected area. In addition, Duranti F et al. (Dermatol Surg., 1998, No. 24 (12), p. 1317-1325) describes a method of injecting hyaluronic acid gel into the affected area. .

 [0003] However, biochemical materials (non-biological tissues) such as those described in each of the above journals are not hydrolyzed in vivo, and the effect of the administration site disappears at an early stage, so the effect does not continue. There is a drawback. Also, it is difficult to completely eliminate immunological effects when using this type of material.

[0004] On the other hand, in the method of injecting the patient's own dermal fibroblasts into the affected area (subcutaneous tissue) after culturing outside the body as described in the above-mentioned International Publication, there is no immunological effect, but it is injected into the affected area Cultured cells function as a filling for the skin tissue (affected area) to be repaired However, it does not have sufficient function to positively improve the physiological state (physical state and Z or nutritional state of the skin tissue) of the skin tissue in the affected area and the surrounding area.

 Disclosure of the invention

 [0005] Therefore, the present invention creates a biocompatible material having a content different from that of a conventional skin tissue repair material (that is, an affected area filling material), and the purpose thereof is skin tissue (affected area) in which an abnormality or defect has occurred. A material for improving skin tissue (composition containing living cells) having a function capable of improving the physiological state of the affected tissue and the surrounding skin tissue to a healthier one with the repair (filling) of It is. Another object of the present invention is to provide a method for improving the physiological state of the affected area by using such a material for improving skin tissue, that is, applying it to the affected area (skin tissue).

 [0006] The skin improving material (that is, a composition for improving skin tissue) provided by the present invention is mainly composed of fibroblasts derived from human or other oral tissues of mammals, And Typically, the skin improvement material (composition for improving skin tissue) disclosed herein comprises a pharmaceutically acceptable medium (including a carrier).

 In this specification, “skin tissue” is a term that should be interpreted broadly unless otherwise specified, and includes epidermis, dermis and subcutaneous tissue.

 In the present specification, “oral tissue” is a term that should be broadly interpreted unless otherwise specified, and includes periodontal tissue (including gingiva, periodontal ligament, alveolar bone, and cementum). )) And vaginal mucosa.

 [0007] The present inventors administered fibroblasts collected from the oral mucosa (for example, gingiva or vaginal mucosa) completely different from the skin tissue to be repaired, to the skin tissue to be repaired. Not only the filling effect on the skin tissue, but also the physiological state of the administration site and its surrounding sites (for example, promoting cell division in the epidermis and the color of the skin based on the skin, galling, texture, etc. Improvement of the physical state of the skin and improvement of the nutritional state of Z or skin), and the present invention has been completed.

In other words, according to the skin tissue improving material (composition for skin tissue improvement) disclosed herein, oral tissue-derived fibroblasts, which are the main constituents of the material, are highly efficient with various cell growth factors ( As a result of continuous production (ie, supply to the affected area) of VEGF, KGF, etc.) at the administration site, the skin tissue (typically subcutaneous tissue) deformation site similar to conventional skin tissue repair materials may be obtained. In addition to exerting a filling effect at the defect site, the physiological state of the site and the surrounding skin tissue can be improved.

 [0008] A preferred embodiment of the skin tissue improving material disclosed herein is a culture obtained by proliferating fibroblasts derived from human or other mammalian oral tissues in vitro as the fibroblasts. It is characterized by being composed mainly of cultured cells.

 Fibroblasts derived from intraoral tissues (preferably periodontal tissue or vaginal mucosa) have a relatively high growth rate in in vitro culture (eg, compared to dermal dermal cells) and a desired amount of intraoral tissue-derived fibers. Blast cells can be produced efficiently in a short period of time. For this reason, according to the skin tissue improving material of this embodiment, the cost for producing fibroblasts can be reduced, and the skin tissue (affected part) can be quickly repaired and improved.

 [0009] In addition, a preferred embodiment of the skin tissue improving material disclosed herein is characterized in that the fibroblasts are human gingival fibroblasts and Z or periodontal ligament fibroblasts. These fibroblasts are particularly active in producing a group of cell growth factors, and can obtain a high skin tissue improvement effect in addition to a skin tissue repair (filling) effect.

 [0010] A preferable skin tissue improving material is prepared in the form of a suspension containing cultured cells obtained by growing the fibroblasts in vitro, and a particularly preferable skin tissue improving material (living cells). A composition containing the cells), wherein the vascular endothelial growth factor (VEGF) content per mg of protein in the supernatant of the suspension is at least 30 pg. It is a skin tissue improving material substantially composed of a liquid. Alternatively, it is substantially a suspension containing the cells, wherein the suspension has a keratinocyte growth factor (KGF) content of at least 0.5 ng per mg of protein in the supernatant of the suspension. It is a skin tissue improving material that is constructed in a general manner. By administering a cell suspension containing such a growth factor at a high concentration (injection into the affected area), the skin tissue can be rapidly improved.

Accordingly, in another aspect of the present invention, gingival fibroblasts and Z or periodontal ligament fibroblasts (preferably in the form of a cell suspension) are administered to the affected area. Methods for supplying VEGF and Z or KGF are provided. Also gingival fibroblasts And a method for producing VEGF and Z or KGF, characterized by culturing Z or periodontal ligament fibroblasts in vitro.

[0011] The present invention also provides a suitable method for producing the skin tissue improving material (composition for improving skin tissue) disclosed herein. That is, the production method of the present invention comprises preparing fibroblasts derived from oral tissues (preferably periodontal tissues) of humans or other mammals, culturing the fibroblasts in vitro, and And collecting the cultured cells. Typically, the cultured cells are suspended in a suitable liquid medium. Preferably, a suspension containing the cultured cells is prepared so that a subject (patient) to which the skin tissue improving material is applied does not contain a substance that can be an immunogen. Further, preferably, fibroblasts derived from oral tissues (preferably periodontal tissues) prepared for culture are collected from a subject (patient) to be applied or a person related to the person. According to the method disclosed here, fibroblasts derived from the oral tissue (preferably periodontal tissue) procured are cultured (proliferated), and a skin tissue improving material having the above-mentioned effects is efficiently produced. Can do.

 [0012] A preferred embodiment of the method for producing a skin tissue improving material disclosed herein is characterized in that the prepared fibroblasts are human gingival fibroblasts and Z or periodontal ligament fibroblasts. In addition, it is particularly preferable that the cells are cultured under conditions capable of producing VEGF and Z or KGF.

 As another aspect, the present invention provides a method for repairing (or improving) the skin tissue of the affected area by injecting the skin tissue improving material disclosed herein into the affected area of the subject.

 This method typically involves a suspension comprising fibroblasts derived from any of the skin tissue improvers disclosed herein (eg, human or other mammalian oral tissues, preferably periodontal tissues). ) And administering (preparing cells) the prepared skin tissue improving material (for example, the above-mentioned fibroblast suspension) to the affected area (skin tissue, for example, subcutaneous tissue) of the subject. To do. Accordingly, the present invention provides a method for using fibroblasts for skin tissue improvement, comprising administering (transplanting) fibroblasts derived from oral tissues of humans or other mammals to affected areas. To do.

[0014] Preferably, human gingival fibroblasts and Z or periodontal ligament fibroblasts are administered to the affected area ( Transplant). Preferably, gingival fibroblasts and Z or periodontal ligament fibroblasts collected from a subject in advance are cultured in vitro, and the resulting self-cultured cells (typically pharmaceutically acceptable with the cultured cells). A cell suspension containing a medium (a suitable solvent) is administered (injected) to the affected area (skin tissue, eg, subcutaneous tissue) of the subject. It is particularly preferred to administer cells cultured under conditions capable of producing VEGF and Z or KGF. Further, it is preferable to use a skin tissue improving material (typically a cell suspension not containing an immunogenic substance) prepared so as not to contain a substance that can be an immunogen. Therefore, the present invention provides a method for improving skin tissue characterized by administering (transplanting) human gingival fibroblasts and Z or periodontal ligament fibroblasts (preferably cells obtained from the patient itself) to the affected area. The method of using the fibroblast is provided.

 By any of the methods disclosed herein, rapid repair and improvement of the affected skin tissue can be realized.

Brief Description of Drawings

FIG. 1 is a graph showing the amount of VEGF produced in the culture medium by human gingival fibroblasts (cultured cells) according to Examples and human skin fibroblasts (cultured cells) according to Comparative Examples. Yes, the horizontal axis is the culture time (h), and the vertical axis is the VEGF concentration (pgZmg protein). The left side of the figure is the result of human gingival fibroblasts (GF), and the right side of the figure is the result of human dermal fibroblasts (DF).

 [Fig. 2] Fig. 2 is a graph showing the amount of KGF produced in the culture solution by human gingival fibroblasts (cultured cells) according to the examples and human skin fibroblasts (cultured cells) according to the comparative examples. Yes, the horizontal axis is the culture time (h), and the vertical axis is the KGF concentration (ngZmg protein). The left side of the figure is the result of human gingival fibroblasts (GF), and the right side of the figure is the result of human skin fibroblasts (DF).

 [FIG. 3] FIG. 3 is a photomicrograph of the tissue after fluorescent immunostaining showing the state of the injection site after 2 months have passed since the human gingival fibroblasts according to the example were injected into the dorsal dermis of nude mice. (Rate 40 times).

[FIG. 4] FIG. 4 is a photomicrograph of the tissue after fluorescent immunostaining showing the state of the injection site after 2 months have passed since the human gingival fibroblasts according to the example were injected into the dorsal dermis of nude mice. (Large rate 400 times).

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0016] Hereinafter, preferred embodiments of the present invention will be described. Note that matters other than matters specifically mentioned in the present specification (for example, the type of fibroblast to be employed) and matters necessary for the practice of the present invention (for example, cell culture, purification method, and cells) General matters such as those relating to the preparation of biological pharmaceutical compositions) are those skilled in the art based on conventional techniques in the fields of medicine, pharmacy, biochemistry, organic chemistry, protein science, molecular biology, veterinary medicine, hygiene, etc. It can be grasped as a design item. The present invention can be carried out based on the contents disclosed in the present specification and common general technical knowledge in the field.

 In addition, the entire contents of all references cited in this specification are incorporated herein by reference.

 [0017] The skin tissue improving material disclosed herein may be a skin tissue improving material (pharmaceutical composition) for humans and other mammals, and oral tissues (preferably periodontium) of humans and other mammals. It is a skin tissue improving material composed mainly of fibroblasts derived from tissue or sputum mucosa. Usually, fibroblasts collected from a subject (subject) that is preferentially used as a component of oral tissues of mammals of the same species as the subject (subject) due to immunological problems Or it is preferable to adopt the cultured cells (autologous cells).

 [0018] As long as it is a fibroblast derived from an oral tissue (preferably periodontal tissue or vaginal mucosa) and can produce various cell growth factors suitable for the purpose of the present invention, the cell type is particularly selected. Although not limited thereto, in the case of targeting humans, gingival fibroblasts of humans (preferably the subject) and Z, or skin tissue improving materials mainly composed of periodontal fibroblasts are preferable. These fibroblasts are preferred as cell materials because they have the ability to efficiently produce various cell growth factors (typically VEGF or KGF) that grow rapidly in vitro (in vitro). ,.

When the cultured cells are used as the main body of the skin tissue improving material, the cultured cells at the stage of actual use are derived from the fibroblasts and the purpose of the present invention (that is, repair and improvement of skin tissue). If the cell growth factor (VEGF, KGF, etc.) required to achieve () is produced, the cell appearance and other factors will be increased by repeated subculture. Even if the nature of the is different from the primary fibroblasts collected. The “fibroblast derived from intraoral tissue” according to the present invention includes cells after such subculture.

 [0019] The method for collecting fibroblasts derived from oral tissues is similar to the conventional method for collecting cells of this type and does not require any special treatment. For example, fibroblasts derived from periodontal tissue (typically gingiva or periodontal ligament) can be obtained from extracted teeth (eg, deciduous teeth and third molars) or extracted gingival tissues and periodontal ligaments. it can.

 In carrying out the present invention, the cells to be used should be collected from the submucosa (for example, the mucous membrane) in the oral cavity! This is an advantage for the target person).

 [0020] The collected fibroblasts can be cultured and propagated in the same manner as the conventional cell culturing method to establish a cell line (subculture system). For example, a general a MEM medium, Eagle medium, Dulbecco's modified Eagle medium (DMEM medium) or the like to which an appropriate serum material, antibiotics or the like are added can be suitably used as the medium. Cultured cells are confluent at about 37 ° C (preferably in a CO incubator) in a suitable culture vessel.

 2

 Incubate until ready, and repeat passage as needed. The number of passages is not particularly limited, but is typically 10 or less (for example, 3 to 6).

[0021] Then, after a desired amount of cultured cells is produced by the culture as described above, the target cells are recovered from the culture vessel. The recovery method is the same as that of the conventional fibroblast, and no special operation / treatment is required for carrying out the present invention. For example, cells attached to the inner wall surface of the culture vessel are treated with an appropriate enzyme (eg, trypsin) to be released and collected together with the culture solution. Preferably, the collected cells are cultured in an appropriate serum-free medium for 12 hours or longer, preferably 24 hours or longer. As a result, immunogenic substances (typically serum components) contained in the medium can be substantially removed. The cultured cells can be cryopreserved by a conventionally known method.

 Thus, a cell suspension from which the immunogenic substance has been substantially removed (ie, a composition based on cultured cells and an appropriate serum-free medium, buffer or other appropriate solvent) is suitable for skin tissue. Can be an improver.

The skin tissue-improving material includes fibroblasts as a main component and a pharmaceutically acceptable medium. In addition to (typically a medium such as a serum-free medium or a solvent such as a buffer), various auxiliary components may be included. For example, (1). Antibiotics, (2). Various vitamins, amino acids, glucose and other nutrient components, (3). Enzymes, (4). Coenzymes, (5). Preservatives, (6) Examples of suitable side components include growth factors such as KGF and VEGF, and other site-in properties, (7) various drugs (for example, anti-inflammatory agents), and (8) pigments.

 [0022] The prepared skin tissue improving material is a conventional skin tissue repair material (see, for example, the above-mentioned patent document (International Publication No. WO97 / 04720) or journal (DeLustro F et al., Duranti F et al.)). Can be administered to the affected area in the same manner. The entire contents of these references are incorporated herein by reference.

 Typically, a skin tissue improving material in the form of a cell suspension can be injected into the subdermal tissue using an appropriate syringe or the like. Moreover, it is not limited to the form of a suspension, and for example, a cell agglomerate composed of a target fibroblast cell may be used as a skin tissue improving material. In this case, it can typically be administered as a complex of fibroblasts and a matrix. As the matrix, various suitable biocompatible materials (polysaccharides, proteins and other high-molecular organic materials, inorganic materials, etc.) can be used. Human-derived (preferably the subject's own) blood or plasma materials of various contents, such as platelet-rich plasma (PRP), are typical examples of suitable Matritas.

 Since the dose and the number of doses (and the interval between doses) can vary depending on the symptoms, there are no particular limitations. Generally, the dose and the number of doses are determined by doctors and other users, and thus do not limit the present invention.

 Moreover, the skin tissue improving material disclosed here can be applied to the improvement of gingival tissue. That is, the present invention provides, as another aspect, fibroblasts derived from oral tissues of humans or other mammals (typically fibroblasts derived from oral tissues of humans or other mammals in vitro). A gingival tissue improving material mainly composed of cultured cells obtained by proliferation in step 1) is provided.

[0023] Several examples relating to the present invention will be described below, but the present invention is not intended to be limited to those shown in the examples.

<Test Example 1: Cultivation of gingival fibroblasts>

Fibroblasts from human gingival tissue were collected. That is, Nagoya University Hospital · Oral Surgery Adult patient power Gingival tissue was collected from the extracted tooth (approved by the Ethics Committee of Nagoya University School of Medicine and obtained the consent of the patient.) O The obtained gingival tissue was 37 ° C for 2 hours. Collagenase treatment (Wako Pure Chemical Industries, Ltd. product: concentration 5 mgZmL) was applied. The treated gingival tissue is transplanted to a culture dish containing a 3.5 cm diameter DMEM medium and cultured under conditions of 37 ° C and 5% CO to obtain fibroblasts (Examples) attached to the dish. It was.

 2

 In addition, normal human adult dermal fibroblasts (product of Cascade Biologics nc., USA) were obtained as target cells (comparative examples).

[0025] Next, DMEM medium (Sigma-) containing 10% ushi fetal serum (Invitrogen product; No. 10099-141) and 1% antibiotic antifungal agent (Invitrogen product; No. 15240-062). Prepare a T75 flask containing Aid rich (No. D6429) and seed approximately 4 x 10 ⁴ adult human gingival fibroblasts or human adult skin (dermis) fibroblasts in the flask. The cells were then cultured for 7 days at 37 ° C and 5% CO (number of tests n = 4). Then, commercially available cell dissociation

 2

 The fibroblasts adhering to the culture flask were recovered by treatment with an agent (Invitrogen product; TrypLE Select (trade name)). The number of collected cells was measured using a commercially available cell counting analyzer (product of Schar fe system; CASY (registered trademark)). The results are shown in Table 1.

 As is clear from the values shown in Table 1, it was confirmed that the growth rate of gingival fibroblasts according to this example was slightly less than twice that of the dermal fibroblasts compared to the target skin fibroblasts. It was.

[0026] [Table 1] Table 1 Cultured cell types Number of seeded cells Number of culture days Number of cells after culture Rate of increase

(Example)

Human gingival fibroblasts 4.0 X 1 0 ⁴ 7 7. 3 4 X 1 0 ⁵ 1 8. 4

 (Comparative example)

Human skin fibroblasts 4.0 X 1 0 ⁴ 7 4. 0 2 X 1 0 ⁵ 1 0. 1

 T—7 5 flask culture results (n = 4)

<Test Example 2: Production of VEGF and KGF by cultured gingival fibroblasts>

10% ushi fetal serum (Invitrogen product; No. 10099-141) and 1% antibiotics The above gingival fibroblasts or dermal fibroblasts are seeded on each well of 6 well plates to which DMEM medium containing a fungicide (Invitrogen product; No. 15240-062) is added, under conditions of 37 ° C and 5% CO. In culture. After starting culture, cultivate in 100% confluent state

2

 The nutrient solution was aspirated and washed twice with PBS, and then DMEM medium without serum was added to continue the cultivation. After 24 hours, 48 hours, and 72 hours, 1 mL of the culture solution was collected, and 1 mL of fresh DMEM medium was added to the well. The culture solution collected at each time was centrifuged for 5 minutes at 4 ° C and lOOOOrpm, and the supernatant was collected. The collected supernatant was stored at -70 ° C as necessary.

 Thus, after collecting all samples, VEGF and KGF in the culture medium were quantified based on ELISA using a commercially available kit (number of tests n = 3). For the quantification of VEGF, “Immunoassay kit VEGF” manufactured by Biosource was used. For quantification of KGF, “Quantikine (registered trademark) Human KGF Immunoassay kit” manufactured by R & D System was used.

[0028] The results are shown in Fig. 1 for VEGF and in Fig. 2 for KGF. In both figures (graphs), the left column graph shows the results for GF (gingival fibroblast), that is, gingival fibroblasts, and the right column graph shows DF (dermal fibroblast), that is, skin fibroblasts. Show the results of the cell.

 As is clear from these graphs, gingival fibroblasts were significantly more capable of producing (secreting) both VEGF and KGF. The amount of VEGF produced by gingival fibroblasts was 30 pg or more per mg of supernatant protein after 24 hours of culture in serum-free DMEM medium. In addition, it was confirmed that about 45 pg and about 60 pg or more of VEGF was produced (secreted) per mg of supernatant protein after 48 hours and 72 hours of culture, respectively. On the other hand, the amount of KGF produced by gingival fibroblasts was 0.5 ng or more (more specifically, 0.8 ng or more) after 1 hour of culture in serum-free DMEM medium. In addition, it was confirmed that about 1.5 ng and about 2 ng or more of KGF was produced (secreted) per mg of supernatant protein after 48 hours and 72 hours of culture, respectively.

[0029] <Test Example 3: Administration of a skin tissue improving material composed of gingival fibroblasts>

Gingival fibroblasts cultured under the same conditions as in Test Example 1 were treated with the cell dissociator and collected from the culture flask. The number of collected cells is determined using the above cell counting analyzer. Measured.

Then, about 2 X 10 ⁷ cells were purchased from a commercially available fluorescent staining kit (Sigma-Aldrich

 The product was stained (fluorescent rabenole) using a PKH26 Red Fluorescent Cell Linker Mini Kit.

Thus, the fluorescently labeled cells were suspended using PBS, and the cell concentration was adjusted to about IX 10 ⁷ cells ZmL, and then injected into the dermis on the back of nude mice.

 Two months later, the injected tissue was collected, and 4% paraformaldehyde solution (Sigma-A ldnch) was used as the fixative.

 The tissue was fixed using a company product. Subsequently, it was embedded in “Tissue-Tek O.C.T. Compound (Sakura Finetechnical Co.)” to prepare a section of about 6 μm.

[0030] Using this section and an anti-human collagen type I monoclonal antibody (Chemicon product), immunostaining was performed by the immunofluorescent antibody method. Subsequently, a commercially available mounting medium for immunohistochemical staining "Vectashield (registered trademark) Mounting Medium for

 Samples were prepared using “Fluorescence with DAPI” (product of Vector Laboratories) and photographed using a microscope (Olympus Corporation product; BX51) and a microscope digital camera (Olympus Corporation product; DP70). The photographs are shown in Figs.

 The blue and fluorescent parts of the photographs shown in Fig. 3 (magnification 40x) and Fig. 4 (magnification 400x) are nuclei stained with the fluorescent dye “DAPI”, and the red! ヽ fluorescent part is the fluorescent dye “PKH26”. Transplanted cells (gingival fibroblasts) stained with “ The green fluorescent site is a fluorescently labeled anti-human collagen type I monoclonal antibody.

 As is clear from these photographic capabilities, the gingival fibroblasts (skin tissue-improving material) transplanted to nude mice remain in the dermis after 2 months, as in the transplantation (ie, (Surviving) was confirmed (Fig. 3). It is also observed that human type I collagen exists in the cytoplasm of the transplanted gingival fibroblasts and the surrounding area. This confirms that gingival fibroblasts still produce type I collagen after transplantation.

 Industrial applicability

[0031] The skin tissue improving material disclosed herein is used to repair skin tissue and improve physiological conditions. It can be performed. For this reason, it is useful as a material used for dermatological (including plastic and cosmetic surgery) treatment.

Claims

The scope of the claims  [1] A skin tissue improving material comprising fibroblasts derived from human or other mammalian oral tissues and a pharmaceutically acceptable medium.  [2] The skin tissue-improving material according to claim 1, wherein the fibroblast includes a cultured cell obtained by proliferating a fibroblast derived from an oral tissue of a human or other mammal in vitro.  3. The skin tissue improving material according to claim 2, wherein the fibroblasts are human gingival fibroblasts and Z or periodontal ligament fibroblasts. [4] The skin tissue improving material according to claim 3, prepared in the form of a suspension containing cultured cells obtained by growing the fibroblasts in vitro. [5] A suspension containing the cells, substantially comprising a suspension having a vascular endothelial growth factor (VEGF) content of at least 30 pg per mg of protein in the supernatant of the suspension. The skin tissue improving material according to claim 4, wherein the skin tissue improving material is constituted. [6] A suspension containing the cells, wherein the suspension has a keratinocyte growth factor (KGF) content of at least 0.5 ng per mg of protein in the supernatant of the suspension. The skin tissue-improving material according to claim 5, which is composed of: [7] A method for producing a skin tissue improving material, comprising:  Providing fibroblasts derived from oral tissues of humans or other mammals; culturing said fibroblasts in vitro; and  Recovering the cultured cells that have been cultured;  Including the method.  [8] The method according to claim 7, wherein the prepared fibroblasts are human gingival fibroblasts and Z or periodontal fibroblasts. [9] The cells are vascular endothelial growth factor (VEGF) and Z or keratinocyte growth factor (KG) The method according to claim 7 or 8, wherein F) is cultured under conditions capable of producing.