Tissue regeneration promotion sheet

Abstract

To provide a tissue regeneration promotion sheet that can stably hold stem cells.SOLUTION: A tissue regeneration promotion sheet 10 comprises a cell retention sheet 12, which is a non-woven fabric that contains fibers mainly composed of polycaprolactone and are capable of allowing permeation of a cell suspension containing stem cells. The fiber diameter of the fibers composing the non-woven fabric may be in the range of 300 nm to 2 μm. The density of the non-woven fabric may be in the range of 1 g / m2 to 3 g / m2. The thickness of the non-woven fabric may be greater than or equal to 300 μm. The tissue regeneration promotion sheet 10 may further comprise a reinforcement sheet 14 that is stacked on the non-woven fabric and allows for permeation of the cell suspension.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a tissue regeneration-promoting sheet. [Background technology]

[0002] In recent years, in the field of regenerative medicine, there has been progress in the development of tissue regeneration promoters that can retain cells inside and promote tissue regeneration by being embedded in the body. For example, Patent Document 1 discloses a sheet-shaped tissue regeneration promoter having at least one of a group consisting of white blood cells and platelets present on its surface. Patent Document 2 discloses a liquid tissue regeneration promoter obtained by mixing a powder of a polymer material with cells. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 4847129 [Patent Document 2] JP 2015-112262 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, the tissue regeneration-promoting materials disclosed in Patent Documents 1 and 2 use powders of porous bodies or polymeric materials, and therefore have the problem that it is difficult to stably retain mesenchymal stem cells, which have a high tissue regeneration ability among cells. Such problems are not limited to the case where mesenchymal stem cells are retained in the tissue regeneration-promoting material, but also exist when other stem cells are retained.

[0005] The present invention has been made against this background, and has an object to provide a tissue regeneration-promoting sheet capable of stably retaining stem cells. [Means for solving the problem]

[0006] In order to achieve the above object, the tissue regeneration-promoting sheet according to the present invention is The nonwoven fabric includes fibers whose main component is polycaprolactone and is capable of being permeated with a cell suspension containing stem cells.

[0007] The fiber diameter of the fibers constituting the nonwoven fabric may be within a range of 300 nm to 2 μm.

[0008] The nonwoven fabric has a basis weight of 1 g / m 2 ~3g / m 2 may be in the range.

[0009] The nonwoven fabric may have a thickness of 300 μm or more.

[0010] The device may further include a reinforcing sheet that is laminated on the nonwoven fabric and is capable of being permeated with the cell suspension.

[0011] The reinforcing sheet may be another nonwoven fabric made of thicker fibers than the fibers constituting the nonwoven fabric.

[0012] The nonwoven fabric may include a first nonwoven fabric and a second nonwoven fabric that is laminated on the first nonwoven fabric and is less prone to deformation than the first nonwoven fabric. Effect of the Invention

[0013] According to the present invention, it is possible to provide a tissue regeneration-promoting sheet capable of stably retaining stem cells. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a perspective view showing the configuration of a tissue regeneration-promoting sheet according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a front view showing a process for producing a tissue regeneration-promoting sheet according to an embodiment of the present invention. [Diagram 3] FIG. 11 is a perspective view showing the configuration of a tissue regeneration-promoting sheet according to a modified example of the present invention. [Figure 4]FIG. 11 is a perspective view showing the configuration of a tissue regeneration-promoting sheet according to another modified example of the present invention. [Diagram 5] FIG. 2 is a diagram showing the capture rate of mesenchymal stem cells by each sample in Example 1. [Figure 6] FIG. 13 is a diagram showing the capture rate of mesenchymal stem cells by each sample in Example 2. [Figure 7] FIG. 13 is a CT image of the rat cervical vertebrae in Example 3. [Figure 8] FIG. 13 shows an immunostained image 6 weeks after bone transplantation in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, a tissue regeneration-promoting sheet according to an embodiment of the present invention will be described in detail with reference to the drawings. In each drawing, the same or equivalent parts are denoted by the same reference numerals.

[0016] The tissue regeneration-promoting sheet according to the embodiment is a sheet that promotes tissue regeneration at a damaged site by being embedded in the damaged site in the body. The tissue regeneration-promoting sheet according to the embodiment includes a nonwoven fabric composed of fine fibers, for example, microfibers, to which stem cells adhere. The nonwoven fabric is a sheet in which fine fibers are intertwined without being woven. Since the nonwoven fabric can be freely deformed, it is suitable for embedding or attaching it to the shape of the damaged site. The tissue regeneration-promoting sheet can be attached or embedded in any organ or organs, for example, the heart, blood vessels, lungs, trachea, esophagus, stomach, small intestine, large intestine, liver, kidneys, bladder, bone, muscle, and skin. The tissue regeneration-promoting sheet can function as a scaffold that promotes the proliferation of specific cells in the body.

[0017] 1, the tissue regeneration-promoting sheet 10 comprises a cell-retaining sheet 12 that retains stem cells, and a pair of reinforcing sheets 14 that are joined so as to sandwich the cell-retaining sheet 12 from above and below and reinforce the cell-retaining sheet 12. The reason for sandwiching the cell-retaining sheet 12 between the pair of reinforcing sheets 14 for reinforcement is that the nonwoven fabric that constitutes the cell-retaining sheet 12 tends to easily curl up when wet due to the properties of the raw materials described below. The cell-retaining sheet 12 and the reinforcing sheet 14 may be joined to each other by an adhesive or thermal fusion, or may be joined by intertwining the fibers during the production of the nonwoven fabric.

[0018] The number of cell-retaining sheets 12 arranged between a pair of reinforcing sheets 14 may be any number within the range in which a cell suspension containing stem cells can be filtered, but it is preferable to stack two or more sheets in order to prevent the cell suspension from leaking from a holder described later when filtering the cell suspension. The cell suspension is a liquid in which cells are suspended in a liquid that can be administered into the body. When multiple cell-retaining sheets 12 are stacked, they may be joined to each other in the same manner as when stacking the cell-retaining sheets 12 and the reinforcing sheets 14. In FIG. 1, the thickness direction of the tissue regeneration-promoting sheet 10 is exaggerated for ease of understanding. The thickness of the tissue regeneration-promoting sheet 10 is set to a degree that allows the cell suspension to be filtered.

[0019] The cell-retaining sheet 12 is a nonwoven fabric containing fibers mainly composed of polycaprolactone. As a result of intensive research by the inventors, it was found that a nonwoven fabric containing fibers mainly composed of polycaprolactone can capture stem cells with high efficiency when filtering a cell suspension containing stem cells, and also promotes the activity of the attached stem cells. Polycaprolactone is a semi-crystalline, biodegradable thermoplastic polyester, and is also suitable for the production of nonwoven fabrics. An example of polycaprolactone is poly-ε-caprolactone. The molecular weight of polycaprolactone may be a molecular weight suitable for injection molding, for example, within the range of 70,000 to 100,000.

[0020] The fibers mainly composed of polycaprolactone may be formed only of polycaprolactone, or may contain other substances. Examples of the other substances include drugs that promote tissue regeneration and other biodegradable polymers. Examples of the drugs that promote tissue regeneration include hydroxyapatite (calcium hydroxide phosphate) and tricalcium phosphate. Examples of the other biodegradable polymers include polylactic acid, polyglycolic acid, polydioxanone, and copolymers thereof.

[0021] The smaller the diameter of the fibers constituting the cell-retaining sheet 12, the higher the stem cell capture rate, but the easier it is for the cell-retaining sheet 12 to curl up when it absorbs moisture. Taking into consideration the stem cell capture rate and ease of handling, the fiber diameter of the fibers constituting the cell-retaining sheet 12 is, for example, within the range of 100 nm to 3 μm, and preferably within the range of 500 nm to 1 μm.

[0022] The larger the basis weight of the cell-retaining sheet 12, the more stem cells can be attached, but the greater the difficulty in filtering the cell suspension. The basis weight of the cell-retaining sheet 12 is set to, for example, 1 g / m2, taking into consideration the amount of attached stem cells and ease of production. 2 ~3g / m 2 Within the range of 1.5 g / m 2 ~2.5g / m 2 It is preferable that the range is 2 g / m 2 It is more preferable that:

[0023] Considering ease of handling with tweezers, the thickness of the cell-retaining sheet 12 is preferably, for example, 300 μm or more, and more preferably 500 μm or more, but it should be noted that the thicker the cell-retaining sheet 12, the more difficult it becomes to filter the cell suspension.

[0024] The stem cells attached to the cell retention sheet 12 are preferably mesenchymal stem cells (MSCs) or cells differentiated from mesenchymal stem cells. Mesenchymal stem cells are somatic stem cells capable of differentiating into cells belonging to the mesenchymal system, and may be derived from any of adipose tissue, periosteum, synovium, cancellous bone, bone marrow, amniotic membrane, umbilical cord blood, and placenta. The stem cells attached to the nonwoven fabric may be, for example, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. Multiple types of stem cells may be attached to the nonwoven fabric together.

[0025] The stem cells to be attached to the cell retention sheet 12 may be collected from a patient and cultured. To culture the stem cells in vitro, for example, a medium containing fetal bovine serum (FBS) or human serum, preferably human serum derived from the patient, or a serum-free medium may be used. The stem cells cultured in the medium may be attached to the nonwoven fabric as is, or the stem cells may be differentiated to express specific cells, such as osteoblasts, and then attached to the nonwoven fabric. To express specific cells, for example, a differentiation inducer may be added to the stem cell medium.

[0026] It is not necessary to attach stem cells to the cell retention sheet 12 in advance. After placing the tissue regeneration promotion sheet 10 at the damaged area inside the body, stem cells can be captured by the cell retention sheet 12 inside the body by administering a cell suspension containing stem cells to the vascular system.

[0027] The reinforcing sheet 14 is a nonwoven fabric that contains fibers mainly composed of a biocompatible polymer and allows a cell suspension to pass through. The reinforcing sheet 14 is bonded to the cell-retaining sheet 12 in order to reinforce the cell-retaining sheet 12, which is easily curled and easily deformed. The biocompatible polymer constituting the reinforcing sheet 14 is a biocompatible polymer stronger than polycaprolactone, for example, a biodegradable polymer such as chitin, chitosan, polylactic acid, or polyglycolic acid, and among these, chitin is preferable. The fiber diameter of the microfiber of the reinforcing sheet 14 is preferably larger than the fiber diameter of the microfiber of the cell-retaining sheet 12. It is not necessary to attach stem cells to the reinforcing sheet 14, but stem cells may be attached to the reinforcing sheet 14 in the process of attaching stem cells to the cell-retaining sheet 12. The above is the configuration of the tissue regeneration-promoting sheet 10.

[0028] Next, a specific procedure for attaching stem cells to the cell-retention sheet 12 according to the embodiment will be described. Stem cells can be attached to the cell-retention sheet 12 by contacting the sheet with a cell suspension containing stem cells for a period of time, but in order to prevent a decrease in the ability to produce growth factors during stem cell culture, the following procedure is preferably used.

[0029] First, as shown in Fig. 2, the cell-retaining sheet 12 and the reinforcing sheet 14, which are stacked on top of each other, are set between a pair of holders 20 constituting the filtration device. Each holder 20 is configured to allow the cell-suspended liquid to flow out or in, and when the cell suspension is flowed from one holder 20 to the other holder 20, the cell-suspended liquid comes into contact with the cell-retaining sheet 12. Note that, although there is a gap between each of the holders in Fig. 2, in reality, the pair of holders 20 are in close contact with each other with the two cell-retaining sheets 12 sandwiched between the pair of reinforcing sheets 14 and joined together.

[0030] Next, the cell suspension containing stem cells is slowly dripped drop by drop from the top, and the cell suspension is filtered through two cell retention sheets 12. The filtration speed is, for example, a rate at which 1 ml of cell suspension is dripped over 2 to 3 minutes. At this time, the stem cells contained in the dripped cell suspension attach themselves to the fibers of the cell retention sheet 12. Although the exact mechanism is unknown, when the cell suspension containing stem cells is filtered little by little through a nonwoven fabric composed of fibers mainly composed of polycaprolactone, the stem cells stably attach to each fiber, and the attached stem cells are activated, promoting the production of growth factors. The above is the specific procedure for attaching stem cells to the cell-retaining sheet 12.

[0031] It should be noted that as long as stem cells are attached to the cell-retaining sheet 12, some production of growth factors from the stem cells can be expected, and therefore the above procedure does not necessarily have to be used.

[0032] As described above, the tissue regeneration-promoting sheet 10 according to the embodiment includes a cell-retaining sheet 12 that contains fibers whose main component is polycaprolactone and that can be permeated with a cell suspension containing stem cells, and a reinforcing sheet 14 that is laminated on the cell-retaining sheet 12 and that can be permeated with a cell suspension. Therefore, the cell-retaining sheet 12 can stably retain stem cells, and the ease of handling by the user can be improved.

[0033] The present invention is not limited to the above-described embodiment, and the following modifications are possible.

[0034] (Modification) In the above embodiment, two cell-retaining sheets 12 are disposed between a pair of reinforcing sheets 14, but this is not limited thereto. The number of cell-retaining sheets 12 sandwiched between the pair of reinforcing sheets 14 may be one, or three or more.

[0035] In the above embodiment, the reinforcing sheet 14 is a nonwoven fabric, but the present invention is not limited to this. For example, the reinforcing sheet 14 may be a porous body that can be permeated with a cell suspension.

[0036] In the above embodiment, the reinforcing sheet 14 is sandwiched between the cell-retaining sheet 12 from above and below, but the present invention is not limited to this. For example, as shown in Fig. 3, the tissue regeneration-promoting sheet 10 may be formed by laminating the reinforcing sheet 14 on one side of the cell-retaining sheet 12. When the strength of the tissue regeneration-promoting sheet 10 can be ensured even if the reinforcing sheet 14 is omitted, or when it is not necessary to ensure the strength of the tissue regeneration-promoting sheet 10, the reinforcing sheet 14 may be omitted. When it is not necessary to ensure the strength of the tissue regeneration-promoting sheet 10, for example, it is when the tissue regeneration-promoting sheet 10 is accommodated in a space within an implant, or when it is rolled up and embedded in a damaged area inside the body.

[0037] In order to ensure the strength of the tissue regeneration-promoting sheet 10 even when the reinforcing sheet 14 is omitted, at least one of the fiber diameter, fiber density, and thickness of the cell-retaining sheet 12 constituting the cell-retaining sheet 12 may be increased, for example. Alternatively, the tissue regeneration-promoting sheet 10 may be formed by laminating a first cell-retaining sheet 12A and a second cell-retaining sheet 12B that is less likely to deform than the first cell-retaining sheet 12A, as shown in FIG. 4. The second cell-retaining sheet 12B may have a larger fiber diameter or a larger fiber density than the first cell-retaining sheet 12A, for example. The first cell-retaining sheet 12A is an example of a first nonwoven fabric, and the second cell-retaining sheet 12B is an example of a second nonwoven fabric that is less likely to deform than the first nonwoven fabric.

[0038] The above-mentioned embodiments are merely examples, and the present invention is not limited to these, and various embodiments are possible without departing from the spirit of the invention described in the claims. The components described in the embodiments and modifications can be freely combined. In addition, inventions equivalent to the inventions described in the claims are also included in the present invention.

[0039] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples. EXAMPLES

[0040] Example 1 In Example 1, a sheet was prepared by laminating a polycaprolactone (PCL) nonwoven fabric and a chitin nonwoven fabric using the method according to the above embodiment, and the capture rate of mesenchymal stem cells in each was evaluated. Specifically, as shown in Figure 1, two PCL nonwoven fabrics were sandwiched between two chitin nonwoven fabrics, one each from above and below, and joined together. The PCL nonwoven fabric had a fiber diameter of 0.5 to 1 μm and a fiber density of 2 g / m2. 2 The chitin nonwoven fabric has a fiber diameter of 8 μm and a fiber density of 30 g / m 2 Next, the PCL nonwoven fabric was sandwiched between a filtration sterilization holder and a cell suspension containing human mesenchymal stem cells was passed through to attach the mesenchymal stem cells to the PCL nonwoven fabric, and the capture rate of the mesenchymal stem cells was evaluated. A Swinex filter holder (Merck Millipore) was used as the holder. As a comparative example, the same experiment was also performed on a sheet in which only two chitin nonwoven fabrics were laminated. The experiment was performed on three samples for each.

[0041] The results are shown in Figure 5. For sheets laminated with chitin nonwoven fabric only, the average cell capture rate was 43.6%, whereas for sheets laminated with PCL nonwoven fabric and chitin nonwoven fabric, the average cell capture rate was 100%. From the above, it was confirmed that PCL nonwoven fabric allows stem cells to attach with high efficiency.

[0042] Example 2 In Example 2, a sheet made by laminating a PCL nonwoven fabric and a polyester (Polyester:PEs) nonwoven fabric, a sheet made of only a PCL nonwoven fabric, and a sheet made by laminating two PCL nonwoven fabrics with different characteristics were prepared, and their handling properties were examined. In addition, a cell suspension was dropped onto each sample, and the cell capture rate was measured.

[0043] When we tried to set a sheet of PCL nonwoven fabric alone in the holder, we found that if the fiber diameter of the PCL nonwoven fabric was 200 μm or less, the PCL nonwoven fabric alone could not be set in the holder. Therefore, we decided to laminate the PCL nonwoven fabric and the PEs nonwoven fabric when the fiber diameter of the PCL nonwoven fabric was 200 μm or less, and to set a sheet of PCL nonwoven fabric alone in the holder when the fiber diameter of the PCL nonwoven fabric was 300 μm or more. In addition, when the fiber diameter was 0.5 μm and the fiber density was 2 g / m 2 , 100μm thick PCL nonwoven fabric, fiber diameter 4.9μm, fiber density 10g / m 2 A sheet was prepared by laminating a PCL nonwoven fabric having a thickness of 500 μm, and this sheet could be set in the holder. The other procedures were the same as in Example 1. Experiments were carried out using three samples for each condition, and the results were as follows: fiber diameter 0.5 μm, fiber density 2 g / m 2 For sheets with a thickness of 100 μm, 2 PEs layers, and 3 to 6 PCL layers, the experiment was performed on only one sample.

[0044] The results are shown in Figure 6. Focusing on the data group with 2 PEs layers, 2 PCL layers, and a thickness of 200 μm, it was found that the thinner the fiber diameter, the higher the cell capture rate. When the fiber diameter was 0.5 μm, the cell capture rate was 100 ± 0.0%, while when the fiber diameter was 2.8 μm, the cell capture rate was 73.4 ± 18.8%. In addition, focusing on the data group with a fiber diameter of 4.9 μm, it was found that the thicker the fiber, the higher the cell capture rate. Specifically, when the thickness was 200 μm, the cell capture rate was 74.9 ± 9.5%, while when the thickness was 500 μm, the cell capture rate was 99.4 ± 3.6%. From the above, it was confirmed that the thinner the fiber diameter and the thicker the fiber, the higher the capture rate of mesenchymal stem cells.

[0045] Example 3 In Example 3, a sheet in which PCL nonwoven fabric and chitin nonwoven fabric were laminated together with bone fragments was implanted into the cervical vertebrae of three immunodeficiency model rats, and bone fusion was evaluated manually 6 weeks after implantation. In addition, bone fusion was also evaluated using CT (Computed Tomography) images of the cervical vertebrae of the immunodeficiency model rats and immunostained images of the collected tissue fragments. The immunostained images were obtained by photographing tissue fragments stained with DAPI (4',6-diamidino-2-phenylindole), a fluorescent dye that binds to DNA (Deoxyribonucleic acid), under a fluorescent microscope. The sheet in which PCL nonwoven fabric and chitin nonwoven fabric were laminated was used with human mesenchymal stem cells attached. As a comparative example, only bone grafting was performed on the cervical vertebrae of five immunodeficiency model rats, and bone fusion was evaluated in the same manner.

[0046] As a result, in the nonwoven fabric combination group, in which a sheet of laminated PCL nonwoven fabric and chitin nonwoven fabric was used in combination, bone fusion was achieved in three out of three mice, resulting in a bone fusion rate of 100%. On the other hand, in the comparative bone graft only group, bone fusion was achieved in only one out of five mice, resulting in a bone fusion rate of 20%. In addition, when bone fusion was evaluated using CT images six weeks after bone grafting, as shown in Figure 7, in the bone graft only group, there was a gap between the bones and bone union had not occurred, but in the nonwoven fabric combination group, bone union was confirmed to have occurred.

[0047] When bone fusion was evaluated using immunostaining images six weeks after bone grafting, it was confirmed that human MSCs had taken root in the rats' bodies in the group in which PCL nonwoven fabric was used. Specifically, cells that were positive for both HNA (Human Nuclear Antigen) and DAPI were human mesenchymal stem cells, and cells that were positive only for DAPI were rat cells. As shown in Figure 8, the image on the right is obtained by superimposing the HNA image on the left and the DAPI image in the middle. When this image is observed, there were many cells that were positive for both HNA and DAPI. Therefore, it was confirmed that bone fusion had occurred even at the cellular level in the group in which PCL nonwoven fabric was used. [Explanation of symbols]

[0048] 10. Tissue regeneration promotion sheet 12 Cell retention sheet 12A First cell-retaining sheet 12B Second cell-retaining sheet 14 Reinforcement sheet 20 Holder

Claims

1. a nonwoven fabric containing fibers whose main component is polycaprolactone and capable of being permeated with a cell suspension containing stem cells; The fiber diameter of the fibers constituting the nonwoven fabric is within the range of 0.3 μm to 4.9 μm, The nonwoven fabric has a basis weight in the range of 6 g / m 2 to 10 g / m 2 . Tissue regeneration promoting sheet.

2. The thickness of the nonwoven fabric is 300 μm or more. The tissue regeneration-promoting sheet according to claim 1.

3. The thickness of the nonwoven fabric is 500 μm or more. The tissue regeneration-promoting sheet according to claim 2.

Images