JP2003325652A - Cell incorporated cultured skin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To increase the take rate of cultured skin, and re-construct a skin structure earlier, particularly, re-produce an epidermis structure. <P>SOLUTION: In this cell incorporated cultured skin, fibroblast and epidermis cornified cell are seeded in a bio absorptive porous base material containing bio absorptive particles for gradually releasing cell growing factors. Thus the rate of reproduction of the skin structure, particularly the formation of the epidermis can be increased. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cultured skin having an epidermis and a dermis layer for regenerating skin tissue to an acute skin defect wound such as burn and trauma and a chronic skin defect wound such as pressure ulcer and ulcer.

[0002]

2. Description of the Related Art When skin defects are widespread, it is necessary to close the skin defects early. As a method of regenerating a defective skin, a method of transplanting a collagen sponge as a template into a living body without using cells (artificial dermis) to regenerate the pseudo dermal tissue in vivo, or culturing only epidermal cells into a sheet There is a method in which only the epidermal layer is regenerated by transplantation (cultured epidermis). In the case of artificial dermis, it is necessary to perform thin split-thickness skin graft or transplant the cultured epidermis after regeneration of dermis-like tissue. Further, since the cultured epidermis does not contain a dermal component, it has a drawback that it is difficult to engraft on a full-thickness skin defect wound in which the dermis layer is also lost.

Therefore, the cultured skin containing the dermis component is Bel
(Bell et al., Science, 211, 1052 (1981)). We have succeeded in producing cultured skin with dermal layer and stratified epidermal layer by inoculating fibroblasts and epidermal cells using two kinds of collagen sponges and then performing air-liquid interface culture (Marguchi Et Plast.Reco
nst. Surg., 93, 537 (1994), Japanese Patent No. 2858066, and Japanese Patent Laid-Open No. 2001-104346). By using this method, it became possible to produce cultured skin in a shorter period of time compared with the conventional method. When autologous cells are used for the cultured skin, there is no rejection reaction, and it can be expected that they will be permanently engrafted. Also,
Since it has both epidermis and dermis, it is not necessary to divide the epidermis into separate layers as in the conventional case, and the presence of epidermal tissue from the early stage of transplantation makes it resistant to external infection.

In order that the cultured skin engraft after transplantation, the adhesion between the cultured skin and the mother bed in the early stage after transplantation is important. That is, on the adhesive surface, invasion of blood vessels from the mother bed is indispensable for supplying nutrients to the transplanted cultured skin. If the nutrient supply is poor, the cells of the transplanted cultured skin will be necrotic, and the transplanted cultured skin itself will fall off. Therefore, after transplantation, it is required to invade blood vessels into cultured skin as soon as possible to supply nutrients and oxygen to cells and enhance adhesion to the mother bed. In addition, cell growth factors or stem cells or progenitor cells that have reached through the blood vessels activate the cells inside the cultured skin to promote regeneration of the skin tissue.

In the case of extensive burns, the collection site of normal skin tissue necessary for preparing cultured skin using autologous cells is limited, and the number of cells obtained per area to be covered with cultured skin is small. turn into. In the case of burns, it is necessary to cover the skin with cultured skin as shortly as possible after injury in order to prevent infection. In such a case, if a method of growing cells in vivo (in-vivo culture method) can be carried out by transplanting the cultured skin in a state where the cell seeding density is low, even in the situation described above, it can be widely used in a short time. It is possible to produce a cultured skin that can cover the skin. Even in this case, it is necessary that the supply of oxygen and nutrients to the cells is sufficient, and for that purpose, it is desired that the microvessels invade the substrate as soon as possible. In addition, early regeneration of not only the dermis but the epidermis is important for enhancing the defense function against infection. These are expected to be promoted by supplying cell growth factors or stem cells, progenitor cells, etc. of biological origin from the regenerated blood vessels.

In order to achieve these objects, cell growth factors having angiogenic activity such as basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (a
FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), plasma version growth factor (PDG)
F), angiopoietin, transforming growth factor (TGF), etc. may be contained in the cultured skin, but these cell growth factors are unstable substances in vivo and only used in an aqueous solution state. Then its biological activity can hardly be expected.

[0007]

DISCLOSURE OF THE INVENTION The present invention aims to increase the engraftment rate of cultured skin and to reconstruct the skin tissue at an early stage, and in particular, to regenerate the epidermal tissue, a cell growth factor is added to the cultured skin substrate. An object of the present invention is to enhance its effect by containing and continuously releasing the cell growth factor.

[0008]

The present invention relates to the following cultured skin. Item 1. A cell-incorporated cultured skin having a dermal layer and an epidermal layer, wherein the dermal layer comprises bioabsorbable particles and fibroblasts for sustained release of cell growth factor in a bioabsorbable first porous substrate. A cell-incorporated cultured skin, wherein the epidermis layer has bioabsorbable particles for sustained release of cell growth factor and epidermal keratinocytes in a bioabsorbable second porous substrate. Item 2. Item 2. The cell-incorporated cultured skin according to Item 1, wherein the bioabsorbable first porous substrate and the second porous substrate are natural polymers. Item 3. Item 2. The cell-incorporated cultured skin according to Item 1, wherein the bioabsorbable particles that release the cell growth factor slowly are made of natural polymers. Item 4. The bioabsorbable first porous substrate has a pore size of 70 to 10
Item 1 comprising a 0 μm collagen sponge and the second porous substrate comprising a collagen sponge having a pore size of 5 to 30 μm
4. The cell-incorporated cultured skin according to any one of 3 to 3. Item 5. Item 5. The cell-incorporated cultured skin according to any one of Items 1 to 4, wherein the bioabsorbable particles that slowly release the cell growth factor are gelatin particles or hyaluronic acid particles having a particle size of 40 µm or less. Item 6. Item 1 in which the cells to be seeded are cells derived from autologous tissue
6. The cell-incorporated cultured skin according to any one of 5 to 5. Item 7. A substrate for preparation of cultured skin, comprising a bioabsorbable first porous base material and a second porous base material which contain bioabsorbable particles that slowly release cell growth factors. Item 8. Biodegradable dermal layer having bioabsorbable particles and fibroblasts in the bioabsorbable first porous substrate, and sustained release of cell growth factor in the bioabsorbable second porous substrate. A method for treating a skin defect part, comprising the step of transplanting a cell-incorporated cultured skin having a bioabsorbable particle and an epidermal layer having epidermal keratinocytes to the skin defect part. Item 9. A cell-incorporated cultured skin comprising a dermis layer having fibroblasts in a bioabsorbable first porous substrate and an epidermal layer having epidermal keratinocytes in a bioabsorbable second porous substrate. A method for treating a skin defect, which comprises the steps of transplanting into a skin defect and introducing bioabsorbable particles that slowly release cell growth factors into the dermis and epidermis.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION First embodiment of bioabsorbability in the present invention
The second porous substrate and the bioabsorbable particles are synthetic polymers or natural polymers obtained by separation / purification from animals, plants, microorganisms and the like, and are polymers that are absorbed in vivo. Specifically, the synthetic polymer is selected from glycolic acid, lactic acid, trimethylene carbonate, homopolymers of polydioxanone, and 2 selected from these.
Examples of the copolymers of the above substances include natural polymers including collagen, gelatin, hyaluronic acid, chondroitin sulfate, alginic acid, agarose, starch, and mixtures thereof. Furthermore, the above-mentioned synthetic polymer and natural polymer can be combined. For seeding and culturing fibroblasts and epidermal cells, collagen is most preferable because of its good cell adhesiveness. Further, it may contain a certain amount of hyaluronic acid.
In addition, gelatin is the most preferable material for the bioabsorbable particles.

The above-mentioned base material is preferably one having a porous structure in order to seed cells and regenerate a tissue three-dimensionally. As having a porous structure,
Nonwoven fabrics, porous films, or sponge-like base materials are conceivable. Since the cultured skin of the present invention regenerates the dermis and the epidermis at the same time, a sponge-like base material having a pore diameter of 70 to 100 μm is used as the first porous base material for seeding fibroblasts, and a second porous base material for seeding epidermal cells is used. The porous substrate has a pore size of 5 to 3
It is preferable to prepare a cultured skin for transplantation by using 0 μm sponge-like base materials and superposing these two kinds of porous base materials and culturing at the liquid-vapor interface.

If the absorption in the body is too fast, the base material will be lost before the tissue is regenerated, and the skin tissue cannot be constructed three-dimensionally. Since it has a bad influence on the regenerated tissue and the living body because it remains, it is possible to produce a porous substrate having a predetermined absorption rate by introducing chemical modification or crosslinking for the absorption period.

The cultured skin of the present invention is produced by seeding fibroblasts and epidermal cells on the above-mentioned porous substrate. Regarding the cells, a stamp-sized tissue is collected from the normal skin of a patient subject to cultured skin transplantation, and epidermal cells and fibroblasts are separated and cultured by a usual method.

The collected cells are seeded on the above-mentioned first or second porous substrate at a seeding density of 1 × 10 ⁴ to 10 ⁷ cells / cm ² and then subjected to a gas-liquid interface culture for a certain time for transplantation. Make cultured skin.

As a cell growth factor, it promotes angiogenesis,
There is no particular limitation as long as it enhances cell activity, and for example, a cell growth factor having an angiogenic action, specifically basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF). , Vascular endothelial growth factor (VE
GF), hepatocyte growth factor (HGF), plasma-derived growth factor (PDGF), angiopoietin, transforming growth factor (TGF). In a particularly preferred embodiment of the invention bFGF was used. bF
It is a well known fact that GF promotes angiogenesis and enhances cell activity, however, bFGF is an unstable substance in the living body, and its expected biological effect even when used in an aqueous solution state. Hardly recognized. Therefore, in producing the cultured skin according to the present invention, it is necessary that the effect of bFGF lasts, and in order to obtain such an effect, it is effective to add the bFGF to a base material for sustained release.

In order to achieve sustained release of bFGF, it is useful to use hydrogel as bioabsorbable particles. Since the bFGF sustained-release carrier hydrogel is uniformly mixed in the complex of the cultured skin sponge and the cells, microspheres are preferable. Examples of the hydrogel material include synthetic polymers such as glycolic acid, lactic acid, trimethylene carbonate, and homopolymers of polydioxanone, and copolymers of two or more substances selected from them. Include collagen, gelatin, hyaluronic acid, chondroitin sulfate, alginic acid, agarose, starch and mixtures thereof. Furthermore, the above-mentioned synthetic polymer and natural polymer can be combined. Other materials used to make hydrogels include polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, poly-2-hydroxyethyl methacrylate, poly
-2-Chemical cross-linked products of synthetic polymers such as hydroxyethyl acrylate, polyacrylamide, polyacrylic acid, polymethacrylic acid and the like, and cross-linked products by radiation irradiation, and further, cross-linked products of copolymers of monomers constituting the above polymers, Cross-linked products of proteins such as dextran, cellulose and albumin and their derivatives can also be used. Particularly preferably, it is effective to use gelatin, which has a proven record in medical use. In this sustained-release system, bFGF is contained and physically immobilized in gelatin microspheres into which cross-linking has been introduced. Immobilized bFGF is water-insolubilized as the gelatin microspheres are decomposed, and as a result, bFGF remains biologically active in this sustained-release system.
Can be released slowly. According to this system, since the sustained release of bFGF completely depends on the degradability of the carrier, the sustained release period can be freely changed even in the case of microspheres having a small diameter.

It has been clarified that the addition of this microsphere to the artificial dermis facilitates the invasion of blood vessels and fibroblasts from surrounding tissues (Kawai.
Biomaterials, 21, 489-499 (2000)). However, invasion of epidermal cells from surrounding tissues is small, and regeneration of skin tissue, particularly epidermal tissue, which can cover a wide area only by this method cannot be expected.

Therefore, in the present invention, fibroblasts and epidermis cells are simultaneously seeded on an absorbable porous substrate, and a cell growth factor is contained therein to release them gradually, thereby producing a regenerated skin tissue having both epidermis and dermis for a short period of time. Can be played back.

[0018]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1 (1) Preparation of Porous Substrate for Fibroblasts 0.5 g of chloroform was added to 50 g of Type I collagen solution diluted to 3 mg / ml, and a homogenizer was used.
What was homogenized at 6,000 rpm for 1 minute was poured into a stainless steel frame and then frozen at -40 ° C, and this was freeze-dried at 30 ° C for 24 hours under vacuum reduced pressure (0.01 mmHg).
Further, it was thermally crosslinked and glutaraldehyde crosslinked, and then freeze-dried again to obtain a collagen sponge having a pore size of 90 μm and a thickness of 3 mm. (2) Preparation of porous culture substrate for epidermal cell culture Type I collagen of 0.3% aqueous solution (pH 3)
Dilute 3 times with 15% ethanol, 0.1% collagen,
It was a 10% ethanol aqueous solution. Furthermore, this solution 15
After pouring g into a petri dish having a diameter of 9 cm and freezing at -135 ° C, the degree of vacuum is 0.1, the drying temperature is 40 ° C, and the drying time is 2
Freeze-dry under the condition of 4 hours, pore size 30μm, thickness 3
mm collagen sponge was obtained. Thereafter, this collagen sponge was sandwiched between a Teflon (R) sheet and a stainless steel plate having a thickness of 3 mm, and a load of 50 kg was applied. Further, in this state, thermal crosslinking was performed under vacuum at 105 ° C. for 24 hours to obtain a porous cell culture substrate for epidermal cell culture. The substrate has a pore size of 5 to 30 μm and a thickness of 40 μm.
It was m. (3) Preparation of gelatin particles 10 wt% of gelatin (isoelectric point 4.9, molecular weight 99,000)
350 ml of 10 ml aqueous solution under stirring (450 rpm) at 40 ° C.
Added in ml of olive oil. After 10 minutes, increase the temperature to 2
The temperature was lowered to 0 ° C., and stirring was continued for another 30 minutes. Acetone 1
00 ml was added and the mixture was stirred at 20 ° C. and 300 rpm.
After 1 hour, using acetone and isopropanol,
The gelatin particles were recovered by centrifugation (5000 rpm) for 5 minutes at 5 ° C. Particles with a particle size of 40 μm or less separated by using a sieve contain 0.1 wt% of Tween 80 2
The particles were chemically crosslinked by stirring in an aqueous solution of wt% glutaraldehyde for 12 hours at 4 ° C. Subsequently, the particles were placed in a 100 mM glycine aqueous solution at 37 ° C. for 3 days.
It was processed for 0 minutes. Glutaraldehyde cross-linked gelatin particles were prepared by centrifuging and separating the particles using a 0.1 wt% Tween 80 solution.

The produced particles were freeze-dried and stored. When the ratio of the dried particle size to the particle size swollen with distilled water was calculated as the water content, the water content was 88.7%. (4) Impregnation with bFGF 2 m of an aqueous solution (12 μl) containing 100 μg of bFGF
g of freeze-dried gelatin particles, and the mixture was allowed to stand at room temperature for 30 minutes for absorption to obtain bFGF-impregnated particles. Empty particles impregnated with an aqueous solution containing no bFGF were also prepared. (5) Culturing of cells Fibroblasts were cultured in a medium containing 10% fetal bovine serum in Dulbecco's Modified Eagle Medium. Epidermal cells were cultured using a serum-free medium (DK-SFM; GIBCO). (6) Preparation of Cultured Skin 1.0 × 10 5 of the cultured fibroblasts was added to a circular collagen sponge having a diameter of 3.5 cm prepared by the method described in (1).
^The seeding was performed so that the seeding density was ⁵ / cm ² . 37 ° C, 5
After culturing in a% CO ₂ incubator for about 6 hours, a 3.5 cm diameter circular epidermal cell culture porous culture substrate prepared by the method described in (2) was placed thereon. Epidermal cells were further seeded thereon at a seeding density of 1.0 × 10 ⁵ / cm ² . Overnight using a serum-free medium for epidermal cells,
Cultured skin was prepared by performing gas-liquid interface culture in a 37 ° C., 5% CO ₂ incubator. (7) Addition of bFGF-containing gelatin particles to cultured skin The addition of bFGF-containing gelatin particles to cultured skin is
12 mg of bFGF-containing gelatin particles and 3 m of physiological saline
1 was added, and syringes were inserted at equal intervals from the inner layer of the cultured skin, and 0.6 ml of each was uniformly injected. (8) Transplantation experiment The back hairs of 6-week-old immunodeficient mice (SCID mice) were removed to prepare a skin defect wound having a diameter of 3.5 cm. Cultured skin prepared by adding the above-mentioned bFGF-containing gelatin particles to this defective wound was sewn after transplantation and protected with a wound dressing. Further, cultured skin to which gelatin particles containing bFGF were not added was similarly transplanted as a control. After 4,7,14 days, the animals were sacrificed and autopsied, and the tissues were fixed with formalin and magnified by a scanning electron microscope, and histological observation (S
EM observation) was performed. <Results> 1) 4 days after transplantation In the system in which bFGF-containing gelatin particles were added, cells were actively invading the cultured skin, and it was confirmed that the cultured skin was well engrafted (Fig. 1). On the other hand, in the control group to which the bFGF-containing gelatin particles were not added, engraftment was observed, but cell invasion was small (FIG. 2). Further, macroscopically, in the group in which the cultured skin containing gelatin particles containing bFGF was transplanted, the surface was already in a dry state, and regeneration of epidermal tissue was remarkably observed (Fig. 3, left). On the other hand, in the control group, the surface was wet and the formation of epidermis was not yet observed (Fig. 3).
right). 2) 7 days after transplantation In the system to which the bFGF-containing gelatin particles were added, the cells continued to invade into the cultured skin, and it was confirmed that the cultured skin was well engrafted (FIG. 4). On the contrary,
Cell invasion also started in the control group to which the bFGF-containing gelatin particles were not added, but it was less than in the system to which the bFGF-containing gelatin particles were added (Fig. 5). 3) 14 days after transplantation In the system in which gelatin particles containing bFGF were added, the epidermal cells were stratified and partly keratinized, and the epidermal tissue was well constructed, and the fibroblasts were also densely spread to the upper part of the cultured skin. The construction of dermal tissue is also good (Fig. 6). On the other hand, in the control group to which gelatin particles containing bFGF were not added, epidermal cells were partially stratified, but they were not keratinized and the epidermal tissue was not sufficiently constructed (FIG. 7).

From the above results, by adding gelatin particles containing bFGF to cultured skin, the dermis from the initial stage of transplantation,
It was revealed that the epidermal tissue was stratified and further keratinized two weeks after the construction of the epidermal tissue was started, and the skin tissue was sufficiently regenerated.

[0021]

EFFECT OF THE INVENTION The combined epidermis / dermis culture skin of the present invention is
The engraftment is good. Further, by forming the dermis layer at an early stage, a reconstructed skin having excellent coordinating property can be constructed and the reproduction of bacteria can be suppressed. In addition, the early formation of the epidermis allows early closure of the wound,
It is effective in preventing bacterial infection from the outside. This is because the conventional cultured skin was only the dermis or only the epidermis, but the cultured skin prepared by the method of the present invention has both epidermis and dermal tissue and can be permanently engrafted by using the patient's own cells. It is possible to prepare various cultured skin.

[Brief description of drawings]

FIG. 1 is a tissue observation 4 days after transplantation of a system containing gelatin particles containing bFGF.

FIG. 2 is a tissue observation 4 days after transplantation of a system in which gelatin particles containing bFGF are not added.

FIG. 3 is a macroscopic observation of the transplanted site 4 days after the transplantation.

[Fig. 4] Tissue observation 7 days after transplantation of a system to which gelatin particles containing bFGF were added.

FIG. 5: Tissue observation 7 days after transplantation of a system in which bFGF-containing gelatin particles are not added.

FIG. 6: Tissue observation 14 days after transplantation of a system in which gelatin particles containing bFGF were added.

FIG. 7: Tissue observation 4 days after transplantation of a system in which bFGF-containing gelatin particles are not added.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shigehiko Suzuki             1 Surpa at 1032 Muroshinmachi, Takamatsu City, Kagawa Prefecture             Su Ritsurin Garden No. 1005 (72) Inventor Yasuhiko Tabata             16 from 3-8 Biwadai, Uji City, Kyoto Prefecture (72) Inventor Kenji Tomihata             No. 1 stone bath, Ikura Shinmachi, Ayabe City, Kyoto             Ze Research and Development Center F-term (reference) 4C081 AB19 BA12 BA13 BA16 BB06                       CD082 CD121 CD152 CD27                       CD29 DB03 EA01

Claims (9)

[Claims] 1. A cell-incorporated cultured skin having a dermis layer and an epidermis layer, wherein the dermis layer gradually releases cell growth factor into a bioabsorbable first porous substrate. Cell-incorporated cultured skin having blast cells, and epidermal layer having bioabsorbable particles for sustained release of cell growth factor and epidermal keratinocytes in a bioabsorbable second porous substrate . 2. The cell-incorporated culture skin according to claim 1, wherein the bioabsorbable first porous substrate and the second porous substrate are natural polymers. 3. The cell-incorporated cultured skin according to claim 1, wherein the bioabsorbable particles that slowly release cell growth factors are made of natural polymers. 4. The bioabsorbable first porous substrate has a pore size of 70.
The cell-incorporated culture skin according to any one of claims 1 to 3, wherein the second porous substrate comprises a collagen sponge having a pore diameter of 5 to 30 µm. 5. The cell-incorporated cultured skin according to any one of claims 1 to 4, wherein the bioabsorbable particles that slowly release the cell growth factor are gelatin particles or hyaluronic acid particles having a particle size of 40 μm or less. 6. The cell-incorporated cultured skin according to claim 1, wherein the cells to be seeded are cells derived from autologous tissue. 7. A substrate for preparation of cultured skin, comprising a bioabsorbable first porous substrate and a second porous substrate that are laminated and that contain bioabsorbable particles that slowly release cell growth factors. 8. A dermal layer having bioabsorbable particles and fibroblasts for sustained release of cell growth factors in a bioabsorbable first porous substrate, cells in a bioabsorbable second porous substrate. A method for treating a skin defect part, which comprises the step of transplanting a cell-incorporated cultured skin having bioabsorbable particles for sustained release of growth factors and an epidermal layer having epidermal keratinocytes to the skin defect part. 9. A cell set comprising a dermal layer having fibroblasts in a first bioabsorbable porous substrate and an epidermal layer having keratinized epidermal cells in a second bioabsorbable porous substrate. A method for treating a skin defect part, which comprises the steps of transplanting embedded culture skin into the skin defect part and introducing bioabsorbable particles that slowly release cell growth factors into the dermis layer and the epidermal layer.