Wound covering material and method for producing the same

Abstract

To provide a wound covering material that can hold cells or microtissue fragments and be simply used for wound healing, and a method for producing the same.SOLUTION: The present invention provides a wound covering material that comprises porous artificial dermis and a lyophilized material containing cells and / or microtissue fragments. The cells and / or microtissue fragments are disposed within the pores of the porous artificial dermis.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a wound dressing and a method for making the wound dressing. [Background technology]

[0002] In wound treatment, regenerative medicine techniques using cultured cells and stem cells have begun to be applied. For example, in the treatment of deep skin defects caused by burns, trauma, bedsores, etc., treatment methods using artificial dermis and autologous cultured epidermis are being applied (e.g., Patent Document 1).

[0003] Autologous cultured epidermis has been used clinically for epidermal reconstruction of extensive burn wounds, but it will not survive if dermal components are not present. Therefore, it is necessary to reconstruct the dermis using artificial dermis or other materials in the graft bed.

[0004] Artificial dermis is made by processing collagen extracted from the skin of cows, pigs, etc. into a sponge-like form. However, there was a problem in that autologous cultured epidermis did not stably attach to skin defect wounds where dermis reconstruction was performed with artificial dermis.

[0005] In response to this, the inventors have demonstrated that by grafting an artificial dermis onto a skin defect wound and adding dedifferentiated fat cells (DFAT), autologous cultured epidermis can be stably engrafted (e.g., Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2016-87187 A Summary of the Invention [Problem to be solved by the invention]

[0007] Attempts have been made to apply cultured cells, microtissue fragments, stem cells, and the like to various wound treatments, including the dedifferentiated fat cells described in Patent Document 1. In order to use cell sheets, cells, or microtissue fragment suspensions for wound treatment, it is necessary to prepare cell sheets and cell suspensions after culturing the cells, and these procedures are cumbersome and require skill.

[0008] Cell sheets and cell suspensions must be frozen when they are preserved. Cryopreservation requires the preparation of freezers, and the frozen state must be maintained when the frozen preserved materials are transported to remote locations. Furthermore, thawing and washing processes are required when the frozen preserved materials are used in clinical settings.

[0009] Furthermore, when a suspension of cells or fine tissue fragments is used to treat a wound, it is difficult to uniformly distribute the cells over the wound, and there is also a problem that the dispersed cells tend to flow out of the wound.

[0010] Therefore, an object of the present invention is to provide a wound dressing that can retain cells or fine tissue fragments and can be easily used for wound treatment, and a method for producing the same. [Means for solving the problem]

[0011] The present invention includes the following aspects. [1] A wound dressing comprising a porous artificial dermis and a freeze-dried material containing cells and / or microscopic tissue fragments, the cells and / or the microscopic tissue fragments being disposed within the pores of the porous artificial dermis. [2] A method for producing a wound dressing, comprising a step of freeze-drying cells and / or fine tissue fragments and a porous artificial dermis to obtain a freeze-dried product, wherein the cells and / or the fine tissue fragments are disposed within the pores of the porous artificial dermis. [3] A method for producing a wound dressing material as described in [2], comprising a step of placing the cells and / or the microtissue fragments in the pores of the porous artificial dermis under reduced pressure prior to the step of obtaining the freeze-dried product. Effect of the Invention

[0012] According to the present invention, it is possible to provide a wound dressing capable of retaining cells or fine tissue fragments and being easily used for wound treatment, and a method for producing the same. [Brief description of the drawings]

[0013] [Figure 1A] 1 is a photograph of a section of artificial dermis impregnated with DFAT cells in Experimental Example 1. [Figure 1B] 1 is a scanning electron microscope image of an artificial dermis impregnated with DFAT cells in Experimental Example 1. [Figure 1C] FIG. 1 shows the results of counting the number of DFAT cells impregnated into an artificial dermis in Experimental Example 1. [Figure 2A] 1 is a scanning electron microscope image of a freeze-dried artificial dermis impregnated with DFAT cells in Experimental Example 2. [Figure 2B] VEGF release amount from artificial dermis impregnated with DFAT cells in Experimental Example 2. [Figure 2C] 1 shows the amount of bFGF released from an artificial dermis impregnated with DFAT cells in Experimental Example 2. [Figure 3A] 1 is a photograph of a section of artificial dermis impregnated with fine tissue pieces in Experimental Example 3. [Figure 3B] 1 is a scanning electron microscope image of an artificial dermis impregnated with fine tissue pieces in Experimental Example 3. [Figure 3C] 1 shows the amount of VEGF released from the artificial dermis impregnated with microtissue pieces in Experimental Example 3. [Figure 3D] 1 shows the amount of bFGF released from the artificial dermis impregnated with micro-tissue pieces in Experimental Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [Wound dressing material] In one embodiment, the present invention provides a wound dressing comprising a porous artificial dermis and a lyophilisate comprising cells and / or microfibers, the cells and / or the microfibers being disposed within the pores of the porous artificial dermis.

[0015] ≪Artificial dermis≫ The artificial dermis is obtained by isolating collagen, the main component of the dermis, from an animal and forming it into a porous (sponge-like) shape. The animal from which the collagen is derived is not particularly limited as long as the effects of the present invention are achieved, but examples thereof include cows and pigs. The collagen constituting the artificial dermis is preferably one that has been treated to reduce antigenicity, such as atelocollagen. The artificial dermis may be one that is commercially available for transplantation, such as PELNAC (registered trademark), TERUDERMIS (registered trademark), and Integra (registered trademark).

[0016] The shape of the artificial dermis is not particularly limited as long as the effects of the present invention are achieved, but it may be, for example, a plate shape, a rectangular plate shape, or a substantially circular plate shape.

[0017] The size of the artificial dermis can be appropriately changed according to the size of the wound to be treated. When the artificial dermis is in the shape of a rectangular plate, the length of the long side of the rectangle may be, for example, 3 mm to 50 cm, or 3 cm to 50 cm. When the artificial dermis is in the shape of a disk, the diameter of the circle may be, for example, 3 mm to 50 cm, or 3 cm to 50 cm.

[0018] The thickness of the artificial dermis is not particularly limited as long as the effects of the present invention are achieved. The thickness may be, for example, 300 μm to 3 cm, more preferably 500 μm to 1 cm, and further preferably 1 to 5 mm.

[0019] The artificial dermis is a porous substrate having a large number of open pores. The number of pores in the artificial dermis is not particularly limited as long as the effects of the present invention are achieved.2 The number of pieces may be 10 to 10,000 per piece.

[0020] The pore size of the artificial dermis may be from 10 μm to 1 mm, preferably from 20 μm to 500 μm, more preferably from 50 μm to 300 μm, further preferably from 50 μm to 200 μm, and particularly preferably from 70 μm to 110 μm. When the diameter of the holes is equal to or greater than the lower limit, the cells or fine tissue fragments can easily enter the holes. When the diameter of the holes is equal to or less than the upper limit, the cells or fine tissue fragments can easily be prevented from flowing out of the holes.

[0021] Cells and / or microscopic tissue pieces are disposed within some or all of the numerous pores that the artificial dermis has.

[0022] ≪Cell≫ The cells that the freeze-dried product may contain are not particularly limited as long as the effects of the present invention are achieved, and may be somatic cells or stem cells.

[0023] The somatic cells may be cells cultured in vitro or in vivo. The cells cultured in vitro may be primary culture cells or established cell lines. The established cell lines are generally immortalized cells and may be cancer cells.

[0024] Examples of somatic cells include fat cells, chondrocytes, osteoblasts, bone cells, muscle cells, vascular endothelial cells, epithelial cells, fibroblasts, nerve cells, hepatic cells, and pancreatic cells. As fat cells, dedifferentiated fat cells are preferable. Somatic cells may be obtained by inducing differentiation from artificial stem cells.

[0025] The stem cells may be stem cells derived from a living organism, or may be artificial stem cells produced by genetic manipulation, etc. Examples of stem cells include embryonic stem cells, tissue stem cells, somatic stem cells, artificial pluripotent stem cells, artificial tissue stem cells, and artificial somatic stem cells.

[0026] The cells may be cells derived from the individual into whom the wound dressing is to be implanted (i.e. autologous cells) or may be cells derived from an individual different to the individual into whom the wound dressing is to be implanted (i.e. allogeneic cells).

[0027] When cells are placed inside the pores of the artificial dermis, the number of cells placed inside each pore may be 1 to 1000, preferably 1 to 100, and more preferably 1 to 10. The number of cells that can be placed in the pores of the artificial dermis is 1 cm 2 Win, 10 2 ~10 8 It may be 10 3 ~10 7 10 is preferred 4 ~10 6 Individual is more preferable.

[0028] ≪Microstructure piece≫ Examples of tissues from which the micro-tissue pieces that the lyophilized product may contain are epithelial tissue, connective tissue, adipose tissue, muscle tissue, nerve tissue, bone tissue, etc. Examples of epithelial tissues include epidermis, etc. Examples of connective tissues include dermis, etc. The micro-tissue pieces may be tissue pieces derived from organoids. Organoids refer to tissues obtained by in vitro three-dimensional culture of tissue stem cells, ES cells, or iPS cells.

[0029] In this specification, a microtissue piece means a tissue piece containing a plurality of cells, and is also called a "micrograft." Here, "a plurality" may be 3 to 1000 cells, preferably 3 to 300 cells, more preferably 3 to 100 cells, and even more preferably 5 to 30 cells.

[0030] The tissue microparticles may be derived from the individual onto whom the wound dressing is to be implanted (i.e. autologous tissue) or may be tissue derived from an individual different to the individual onto whom the wound dressing is to be implanted (i.e. allogeneic tissue).

[0031] The tissue microparticle may contain biological material other than cells, such as extracellular matrix.

[0032] The size of the fine tissue pieces is not particularly limited as long as the effects of the present invention are achieved, but may be, for example, 10 to 300 μm, preferably 20 to 100 μm, and more preferably 35 to 50 μm.

[0033] When microfine tissue pieces are disposed inside the pores, the number of microfine tissue pieces inside one pore may be 1 to 100, or may be 1 to 10. The number of micro-tissue pieces that can be placed in the pores of the artificial dermis is, for example, 1 cm of artificial dermis. 2 Win, 10 2 ~10 4 It may be one piece.

[0034] The water content of the lyophilized product may be, for example, 20% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less, 0.3% by mass or less, or 0.1% by mass or less, based on the total mass (100% by mass) of the lyophilized product. The lower limit of the water content is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 0.01% by mass, or may be 0% by mass measured.

[0035] The porous artificial dermis and the freeze-dried material containing cells and / or fine tissue fragments can be produced by the method described below.

[0036] The wound dressing according to the present embodiment may include other components in addition to the freeze-dried material, such as a dried or freeze-dried product of a bioabsorbable polymer, such as polylactic acid or polyalkylene glycols, or a hydrogel, such as gelatin.

[0037] <Transplantation Target> The subject of transplantation of the wound dressing according to the present embodiment is not particularly limited as long as the effect of the present invention is achieved, and is preferably a mammal. Examples of mammals include rodents such as rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. Among these, humans are preferred.

[0038] The transplantation site of the wound dressing according to the present embodiment is not particularly limited as long as the effects of the present invention are achieved, and may be a wound. Examples of tissues and organs that may be wounds include skin, muscle, bone, brain, liver, heart, spleen, lung, kidney, pancreas, stomach, intestines, and reproductive organs, and among these, skin is preferred.

[0039] The cells and / or fine tissue fragments contained in the lyophilized material are preferably those present in the organ at the transplantation site. For example, when the wound dressing is transplanted to a skin wound, the lyophilized material is preferably cells and / or fine tissue fragments obtained from the skin, cells having characteristics similar to those of cells present in the skin and artificially produced by genetic manipulation or the like.

[0040] Alternatively, the cells and / or tissue fragments contained in the freeze-dried material preferably have a biomolecule that enhances the wound healing effect, or can release the biomolecule. Examples of the biomolecule include VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), EGF (epidermal growth factor), PDGF (platelet derived growth factor), etc. Examples of cells that have such a biomolecule or can release the biomolecule include epidermal cells, fibroblasts, dedifferentiated adipocytes, stem cells, etc.

[0041] The wound dressing according to the present embodiment described above can hold cells or microscopic tissue pieces and can be easily used for wound treatment. The artificial dermis and the cells and / or microscopic tissue pieces contained in the wound dressing are freeze-dried. Therefore, this wound dressing can be stored at room temperature. No cooling or freezing equipment is required for storage of the wound dressing, and it can be easily transported.

[0042] Since the cells and / or microscopic tissue fragments are disposed in the pores of the artificial dermis, the cells and / or microscopic tissue fragments are retained in the artificial dermis, which makes it easier to prevent the cells and / or microscopic tissue fragments from flowing out of the wound when the wound dressing is grafted onto the wound, and makes it easier to control the amount of the cells and / or microscopic tissue fragments in the wound.

[0043] The cells and / or fine tissue fragments contained in the lyophilized material can release growth factors such as VEGF, bFGF, etc. to the same extent as or greater than cells and / or fine tissue fragments that have not been lyophilized. Therefore, by grafting the wound dressing onto the wound, it is possible to enhance the therapeutic effect.

[0044] The cells and / or tissue fragments contained in the wound dressing are freeze-dried to eliminate their immunogenicity, thereby preventing rejection of allogeneic cells, tissue or cultures thereof.

[0045] [Manufacturing method of wound dressing] In one embodiment, the present invention provides a method for producing a wound dressing. The wound dressing according to the embodiment can be produced by the method according to the embodiment. The method for producing a wound dressing according to the embodiment includes the following steps (A) and (B).

[0046] Process (A): Placing the cells and / or micro-tissue fragments into the pores of the porous artificial dermis under reduced pressure. Process (B): freeze-drying the porous artificial dermis with the cells and / or the micro-tissue pieces disposed within the pores;

[0047] (Process (A)) In step (A), cells and / or fine tissue fragments are placed in the pores of the porous artificial dermis under reduced pressure. Under reduced pressure, air in the pores of the artificial dermis is sucked, and the cells and / or fine tissue fragments are easily placed in the pores of the artificial dermis. "Placing the cells and / or fine tissue fragments in the pores of the artificial dermis" can also be expressed as "impregnating the artificial dermis with the cells and / or fine tissue fragments."

[0048] The reduced pressure may be, for example, 90 kPa or less, 70 kPa or less, 50 kPa or less, 30 kPa or less, or 10 kPa or less. The lower limit of the reduced pressure is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 1 kPa or 0 Pa in terms of measurement.

[0049] In step (A), the time for impregnating the cells and / or fine tissue pieces into the artificial dermis may be from 1 second to 1 hour, preferably from 10 seconds to 10 minutes, and more preferably from 1 to 3 minutes.

[0050] The temperature conditions in step (A) are preferably 0°C to 45°C, more preferably 10°C to 45°C, and even more preferably 20°C to 40°C.

[0051] (Process (B)) In step (B), the artificial dermis obtained in step (A), in which the cells and / or the microscopic tissue pieces are disposed within the pores, is freeze-dried.

[0052] The temperature conditions for freeze-drying are preferably -200°C to -5°C, more preferably -100°C to -10°C, and even more preferably -50°C to -20°C.

[0053] The atmosphere in the freeze-drying is preferably a reduced pressure. The reduced pressure may be, for example, 1 kPa or less, 500 Pa or less, 300 Pa or less, 200 Pa or less, 100 Pa or less, 50 Pa or less, 30 Pa or less, or 20 Pa or less. The lower limit of the reduced pressure is not particularly limited as long as the effects of the present invention are achieved, and may be, for example, 1 Pa or 0 Pa in terms of measurement.

[0054] In step (B), the time for freeze-drying may be from 10 minutes to 48 hours, and is preferably from 5 hours to 30 hours.

[0055] The equipment used for freeze-drying is not particularly limited as long as the effects of the present invention are achieved, and any known equipment can be used.

[0056] The artificial dermis may be the same as those described above in the previous embodiment. The cells and fine tissue pieces include the same as those mentioned above in the previous embodiment.

[0057] The lyophilized product obtained in step (B) may be used as a wound dressing as it is, or may be appropriately combined with other materials to produce a wound dressing. EXAMPLES

[0058] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0059] [Experimental Example 1] According to the procedure described below, human DFAT cells (Dedifferentiated fat cells; DFAT) were impregnated into the artificial dermis under reduced pressure.

[0060] PELNAC (registered trademark, manufactured by GUNZE) was used as the artificial dermis. The size of the artificial dermis was 1 cm × 1 cm, and the thickness was 3 mm. A simple pickling machine (manufactured by ZOJIRUSHI) was used as the impregnation device. Cultured human DFAT cells were suspended in physiological saline to obtain a cell suspension.

[0061] Next, the cell suspension was contacted with one side of the artificial dermis. 1 × 10 human DFAT cells were placed on the artificial dermis. 6 In control group 1, the DFAT cells were brought into contact with the artificial dermis and then allowed to stand for 2 minutes at atmospheric pressure (101 kPa) without reducing the pressure. In experimental group 2, the pressure was reduced to 88 kPa and the cells were allowed to stand for 2 minutes under this reduced pressure. In experimental group 3, the pressure was reduced to 8 kPa and the cells were allowed to stand for 2 minutes under this reduced pressure. These operations were carried out at room temperature (23°C). Next, for control group 1 and experimental group 2, the artificial dermis containing the DFAT cells was fixed with formalin.

[0062] The artificial dermis after fixation obtained in control group 1 and experimental group 2 was embedded in paraffin and sectioned. The sections were then subjected to hematoxylin-eosin staining. The results are shown in Figure 1A. In Figure 1A, the arrow indicates the DFAT cells impregnated in the artificial dermis. It was confirmed that in control group 1, the DFAT cells were not impregnated in the artificial dermis, but in experimental group 2, the DFAT cells were impregnated in the artificial dermis. In other words, it was confirmed that in control group 1, the DFAT cells were not placed in the pores of the artificial dermis, but in experimental group 2, the DFAT cells were placed in the pores of the artificial dermis.

[0063] The artificial dermis impregnated with DFAT cells obtained in experimental group 2 was observed with a scanning electron microscope. The results are shown in Figure 1B. From these results, it was confirmed that DFAT cells were placed in the pores of the artificial dermis.

[0064] The sections of the artificial dermis obtained in experimental groups 2 and 3 were divided into one surface (upper layer) that had been contacted with DFAT cells and the opposite surface (lower layer), and the number of DFAT cells impregnated per unit area (210 × 210 μm) was counted in each section. The results are shown in Figure 1C.

[0065] 1 In experimental group 2 (reduced pressure of 88 kPa), the number of cells in the lower slice was smaller than the number of cells in the upper slice. In experimental group 3 (reduced pressure of 8 kPa), the number of cells in the lower slice was similar to the number of cells in the upper slice. The number of cells in the lower slice in experimental group 3 was significantly greater than the number of cells in the lower slice in experimental group 2 (p<0.05, Tukey-Kramer method). It was confirmed that DFAT cells could be impregnated into the entire thickness of the artificial dermis by reducing the pressure to 8 kPa and impregnating the DFAT cells.

[0066] [Experimental Example 2] According to the following procedure, human DFAT cells were impregnated into an artificial dermis under reduced pressure, and then the resultant was cryopreserved or vacuum freeze-dried. The amount of growth factor released from the obtained sample was measured.

[0067] First, 1 × 10 human DFAT cells were added to the same artificial dermis (size 1 cm × 1 cm, thickness 3 mm) as used in Experimental Example 1. 6 The artificial dermis was contacted with the DFAT cells, the pressure was reduced to 8 kPa, and the artificial dermis was left to stand under the reduced pressure for 2 minutes to impregnate the DFAT cells. Next, in experimental group 2, the artificial dermis impregnated with the cells was frozen at -80°C for 1 week. In experimental group 3, the artificial dermis impregnated with the cells was vacuum freeze-dried. In experimental group 3, a vacuum freeze-dryer (CHList, ALPHA 1-4 / 2-4 LSCplus, manufactured by KUBOTA) was used. In experimental group 3, pre-freezing was performed at -35°C for 30 minutes. Then, vacuum freeze-drying was performed for about 26 hours in the following order: -35°C for 10 minutes, -20°C for 10 hours, -15°C for 4 hours, -10°C for 2 hours, 0°C for 2 hours, 10°C for 3 hours, and 20°C for 4 hours. The pressure for vacuum freeze-drying was 10 Pa. In control group 1, the cell-impregnated artificial dermis was not treated.

[0068] The freeze-dried product obtained in experimental group 3 was observed with a scanning electron microscope. The results are shown in Figure 2A. In Figure 2A, the arrow indicates the DFAT cells impregnated in the artificial dermis. It was confirmed that the DFAT cells were arranged within the pores of the artificial dermis in the freeze-dried product.

[0069] Next, the samples of experimental groups 2 to 3 and control group 1 were immersed in 2 mL of physiological saline containing 1% FBS and stirred. The amount of VEGF and bFGF released in this liquid was measured by ELISA. VEGF was measured using Quantikine (R&D systems, Inc., Cat. No. DVEOO), and bFGF was measured using Quantikine (R&D systems, Inc., Cat. No. DFB50). The results are shown in Figures 2B to C. Note that VEGF and bFGF were not detected in the 2 mL of physiological saline containing 1% FBS in which the samples were not immersed.

[0070] FIG. 2B shows the results of measuring the amount of VEGF released. FIG. 2C shows the results of measuring the amount of bFGF released. From the results shown in FIG. 2B, it was confirmed that VEGF was released to the same extent in experimental group 2 (cryopreserved group) and experimental group 3 (vacuum freeze-dried group) as in control group 1 (untreated group). From the results shown in FIG. 2C, it was confirmed that more VEGF was released in experimental group 3 (vacuum freeze-dried group) than in control group 1 (untreated group) and experimental group 2 (cryopreserved group).

[0071] These results indicate that the artificial dermis impregnated with DFAT cells releases growth factors to the same extent or even more than the untreated case, even after freeze-drying.

[0072] [Experimental Example 3] According to the procedure described below, fine tissue pieces of rat epidermis and dermis were impregnated into the same artificial dermis (size 1 cm × 1 cm, thickness 3 mm) as the artificial dermis used in Experimental Example 1, and then the artificial dermis was frozen and stored or vacuum freeze-dried. The amount of growth factor released from the obtained samples was measured.

[0073] First, small pieces of epidermis (s) and dermis (d) measuring approximately 2 mm were excised from a rat, and micro-tissue pieces measuring 35 to 50 μm were prepared using RIGENERA (Human Brain Wave, Inc.). These micro-tissue pieces were suspended in 1 mL of physiological saline.

[0074] Next, the suspension of each microtissue fragment was brought into contact with the artificial dermis, the pressure was reduced to 48 kPa, and the artificial dermis was allowed to stand for 2 minutes under the reduced pressure to impregnate each microtissue fragment into the artificial dermis. In experimental groups 1s, 2s, and 3s, the artificial dermis was impregnated with microfine pieces of epidermis (s). In experimental groups 1d, 2d, and 3d, the artificial dermis was impregnated with microfine pieces of dermis (d).

[0075] The artificial dermis obtained after fixation in the control group 1d and the experimental group 2d was embedded in paraffin and sectioned. The sections were then subjected to hematoxylin-eosin staining. The results are shown in Figure 3A. In Figure 3A, the arrow indicates the dermal micro-tissue fragments impregnated in the artificial dermis. It was confirmed that in the control group 1d, the dermal micro-tissue fragments were not impregnated in the artificial dermis, but in the experimental group 2d, the dermal micro-tissue fragments were impregnated in the artificial dermis. In other words, it was confirmed that in the control group 1d, the dermal micro-tissue fragments were not placed in the pores of the artificial dermis, but in the experimental group 2d, the dermal micro-tissue fragments were placed in the pores of the artificial dermis.

[0076] The artificial dermis obtained in experimental group 2d after fixation was photographed with a scanning electron microscope. The results are shown in Figure 3B. In Figure 3B, the arrow indicates the dermal microtissue piece impregnated in the artificial dermis. It was confirmed that the dermal microtissue piece impregnated in the artificial dermis maintained its shape.

[0077] Next, in experimental groups 2s and 2d, the artificial dermis impregnated with each micro-tissue fragment was frozen. In experimental groups 3s and 3d, the artificial dermis impregnated with each micro-tissue fragment was vacuum freeze-dried. Freezing and vacuum freeze-drying were performed in the same manner as in experimental example 2. In control groups 1s and 1d, the artificial dermis impregnated with each micro-tissue fragment was not treated.

[0078] Next, the amounts of VEGF and bFGF released were measured by ELISA for the samples of experimental groups 2 and 3. The amounts of VEGF and bFGF released were measured in the same manner as in Experimental Example 2. The results are shown in Figures 3C and 3D. Figure 3C shows the results of measuring the amount of VEGF released. Figure 3D shows the results of measuring the amount of bFGF released.

[0079] The results shown in FIG. 3C confirmed that VEGF was released in experimental group 3s to the same extent as in control group 1s, and that VEGF was released in experimental group 3d to the same extent as in control group 1d. The results shown in FIG. 3D confirmed that bFGF was released in experimental group 3s to the same extent as in control group 1s, and that bFGF was released in experimental group 3d to the same extent as in control group 1d. In experimental groups 3s and 3d, it was confirmed that approximately 10 times the amount of bFGF was released compared to when DFAT cells were impregnated into the artificial dermis. [Industrial Applicability]

[0080] According to the present invention, it is possible to provide a wound dressing that can hold cells or microscopic tissue pieces and can be easily used for wound treatment, and a method for producing the same. The present invention can be suitably used for wound treatment.

Claims

1. The present invention relates to a method for producing a lyophilized material comprising the steps of: A wound dressing, wherein the cells and / or the micro-tissue fragments are disposed within the pores of the porous artificial dermis.

2. The method includes a step of freeze-drying the cells and / or microtissue pieces and the porous artificial dermis to obtain a freeze-dried product, The cells and / or the microtissue fragments are disposed within the pores of the porous artificial dermis. A method for manufacturing a wound dressing.

3. Prior to the step of obtaining the freeze-dried product, The method for producing a wound dressing according to claim 2, further comprising the step of disposing the cells and / or the microfine tissue fragments in the pores of the porous artificial dermis under reduced pressure.

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