Tissue regeneration implant, and method for manufacturing a tissue regeneration implant.

Abstract

The present invention provides a tissue regeneration implant that exhibits good tissue regeneration and implant replacement in the recipient, and a method for manufacturing the tissue regeneration implant. [Solution] A tissue regeneration implant for implantation in a living body, comprising an implant, cells supported by the implant, and fibrin supported by the implant. A method for manufacturing a tissue regeneration implant for implantation in a living body, comprising a cell impregnation step of impregnating an implant with cells, and a fibrin impregnation step of impregnating the implant, which is supported by the cells, with fibrin after the cell impregnation step.

Description

Technical Field

[0001] The present invention relates to a tissue regeneration implant to be implanted in a living body for inducing tissue regeneration, and a method for producing the tissue regeneration implant.

Background Art

[0002] Implants are used for implantation into living bodies to replace tissues lost due to diseases or damage. However, since living bodies maintain their homeostasis by repeatedly undergoing destruction and regeneration, it is preferable for an implant to have the function of being replaced by endogenous substances in the living body, rather than remaining permanently after treatment. In fact, it has been reported that the treatment success rate is low when artificial implants are used (Non-Patent Document 1).

[0003] In tissue replacement surgery using implants, implants made of bioabsorbable materials are also occasionally found. Said bioabsorbable materials promote tissue regeneration before the implanted implant is biodegraded. However, in said tissue replacement surgery, when the replacement volume exceeds a certain level, the artificial material is removed in a situation where tissue regeneration in the living body is insufficient. For this reason, it has been suggested that there is a possibility that the treatment may not succeed. In particular, when using an implant for the treatment of structures in joints, both strength and a repair mechanism are required, and thus various contrivances have been made to induce tissue regeneration on artificial materials (for example, Patent Documents 1 to 5).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

[0005] [Non-Patent Document 1] Wojciech Satora et al., Polim Med, 2017, 47(1), p.55-59. [Overview of the project] [Problems that the invention aims to solve]

[0006] However, there is still room for improvement in the methods used to induce organizational restructuring. This invention has been made in view of the above circumstances, and aims to provide a tissue regeneration implant that exhibits good tissue regeneration and implant replacement in the recipient, and a method for manufacturing the tissue regeneration implant. [Means for solving the problem]

[0007] The inventors of this invention have discovered that by loading cells and fibrin onto the implant, the implant can be replaced by the recipient's cells early after transplantation, thus completing the present invention.

[0008] In other words, the present invention includes the following embodiments. [1] A tissue regeneration implant for implantation in a living organism, comprising an implant, cells supported on the implant, and fibrin supported on the implant. [2] The tissue regeneration implant according to [1], wherein the implant is a collagen sponge. [3] A tissue regeneration implant described in [1] or [2], which is implanted within the meniscus. [4] The tissue regeneration implant according to any one of [1] to [3], wherein the fibrin is fibrin injected into the implant after the implant carrying the cells has been transplanted into the living body. [5] The tissue regeneration implant according to any one of [1] to [4], wherein the fibrin is produced by injecting a fibrinogen solution and a thrombin solution into the implant carrying cells and allowing them to react within the implant. [6] The tissue regeneration implant according to any one of [1] to [5], wherein the cells are cartilage cells. [7] The tissue regeneration implant according to [6], wherein the chondrocytes are infant-derived chondrocytes. [8] 3.0 × 10 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3 A tissue regeneration implant according to any one of [1] to [7], containing the following cells. [9] A method for manufacturing a tissue regeneration implant to be implanted in a living body, comprising: a cell impregnation step of impregnating an implant with cells; and a fibrin impregnation step of impregnating the implant carrying the cells with fibrin after the cell impregnation step.

[10] The method for manufacturing a tissue regeneration implant according to [9], wherein the implant is a collagen sponge.

[11] A method for manufacturing a tissue regeneration implant according to [9] or

[10] , comprising a transplantation step of transplanting the implant carrying the cells into the living body after the cell impregnation step and before the fibrin impregnation step.

[12] The method for manufacturing a tissue regeneration implant according to any one of [9] to

[11] , wherein the biological tissue is within the meniscus.

[13] A method for producing a tissue regeneration implant according to any one of [9] to

[12] , wherein in the fibrin impregnation step, a fibrinogen solution and a thrombin solution are injected into the implant carrying the cells and reacted within the implant to generate fibrin.

[14] A method for manufacturing a tissue regeneration implant according to any one of [9] to

[13] , wherein the cells are cartilage cells.

[15] The method for producing a tissue regeneration implant according to

[14] , wherein the chondrocytes are infant-derived chondrocytes.

[16] In the cell impregnation step, 3.0 × 10 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3 A method for manufacturing a tissue regeneration implant according to any one of [9] to

[15] , wherein the implant is supported by the following cells. [Effects of the Invention]

[0009] The present invention provides a tissue regeneration implant that exhibits good tissue regeneration and implant replacement in the recipient, and a method for manufacturing the tissue regeneration implant. [Brief explanation of the drawing]

[0010] [Figure 1A] Figure 1A shows the expression ratio of type I collagen (Col1 in the figure) to GAPDH in cells cultured for 1, 2, or 4 weeks (1W, 2W, 4W in the figure) in a collagen sponge that supports cells (hereinafter also simply referred to as "cell-supported collagen sponge") (F(-) in the figure) or a collagen sponge that supports both cells and fibrin (hereinafter also simply referred to as "cell and fibrin-supported collagen sponge") (F(+) in the figure) (Test Example 1). [Figure 1B] Figure 1B shows the expression ratio of ACAN to GAPDH in cells cultured for 1, 2, or 4 weeks (1W, 2W, 4W) in a cell-supported collagen sponge (F(-) in the figure) or a cell and fibrin-supported collagen sponge (F(+) in the figure) (Test Example 1). [Figure 2A] Figure 2A shows the expression ratio of type I collagen (Col1 in the figure) to GAPDH in cells after culturing for 1, 2, or 4 weeks (1W, 2W, 4W in the figure) in either a low-concentration group of cells and fibrin-carrying collagen sponge (L) (L in the figure) or a standard group of cells and fibrin-carrying collagen sponge (S) (S in the figure) (Test Example 2). [Figure 2B] Figure 2B shows the expression ratio of ACAN to GAPDH in cells after culturing for 1, 2, or 4 weeks (1W, 2W, 4W in the figure) in either a low-concentration group of cells and fibrin-carrying collagen sponge (L) (L in the figure) or a standard group of cells and fibrin-carrying collagen sponge (S) (S in the figure) (Test Example 2). [Figure 3] The upper panel of Figure 3 shows FISH images (from left to right: cell nucleus, porcine cells, human infant-derived chondrocytes, and their merge) of porcine menisci with cell-carrying collagen sponges embedded in them, from Test Example 3, after two weeks of culture. The lower panel of Figure 3 shows FISH images (from left to right: cell nucleus, porcine cells, human infant-derived chondrocytes, and their merge) of porcine menisci with cell-carrying and fibrin-carrying collagen sponges embedded in them, from Test Example 3, after two weeks of culture. [Figure 4] The upper panel of Figure 4 shows FISH images (from left to right: cell nucleus, porcine cells, human infant-derived chondrocytes, and their merge) of porcine menisci with cell-supported collagen sponges embedded in them, cultured for 4 weeks, in Test Example 3. The lower panel of Figure 4 shows FISH images (from left to right: cell nucleus, porcine cells, human infant-derived chondrocytes, and their merge) of porcine menisci with cell-supported and fibrin-supported collagen sponges embedded in them, cultured for 4 weeks, in Test Example 3. [Figure 5A] Figure 5A shows the expression ratio of PD-L1 to GAPDH in cells cultured for 3 days in a collagen sponge carrying adult-derived chondrocytes or a collagen sponge carrying human infant-derived chondrocytes (Test Example 4). [Figure 5B] Figure 5B shows the expression ratio of PD-L2 to GAPDH in cells cultured for 3 days in a collagen sponge carrying adult-derived chondrocytes or a collagen sponge carrying human infant-derived chondrocytes (Test Example 4). [Figure 6]Figure 6 shows the expression ratios of type I collagen (COL1A1), type II collagen (COL2A1), MMP3, MMP13, RUNX2, and type X collagen (COL10A1) to GAPDH in human infant-derived chondrocytes cultured on cell-supported collagen sponges at 0.9 × 10⁵ cells (LD group), 1.8 × 10⁵ cells (MD group), and 3.6 × 10⁵ cells (HD group) (Test Example 5). In the figure, * means p < 0.05, ** means p < 0.01, and **** means p < 0.0001. [Figure 7] Figure 7 shows the results of the mRNA expression analysis of collagen sponges supported by infant-derived chondrocytes (PDCs) and adult-derived chondrocytes (CDCs) or adult-derived synovial cells (SDCs) (Test Example 6). [Modes for carrying out the invention]

[0011] <Tissue regeneration implants> The tissue regeneration implant of this embodiment is implanted in a living body and comprises an implant, cells supported on the implant, and fibrin supported on the implant.

[0012] In the tissue regeneration implant of this embodiment, "in vivo" means the inside of an animal. Examples of such animals include mammals such as humans, dogs, cattle, pigs, goats, and sheep, as well as birds, fish, and other animals. Examples of the inside of the animal include subcutaneous tissue in the limbs, lumbar region, back, and abdomen, as well as the abdominal cavity. Within the living body, the limbs of humans are preferred, and the joints of humans are more preferred. Examples of such joints include the ligaments, menisci, cartilage, and bones. Among these joints, the meniscus is preferred.

[0013] In the tissue regeneration implant of this embodiment, transplantation means placing an extracorporeal substance into the living body for the purpose of transferring and implanting tissue or organs into the living body. The tissue or organ to be transferred may be the whole or a part of it. In transplantation, the tissue regeneration implant may be directly implanted into the living body, or it may be implanted using a bioadhesive or sutures.

[0014] The implant in this embodiment of tissue regeneration is a structure molded with the premise of being transplanted into the living body. In particular, it is preferable that it be composed of a biodegradable polymer that decomposes in the living body. Examples of the above-mentioned biodegradable polymers include polylactic acid, polycaprolactone, polybutylene succinate, polyethylene succinate, polyvinyl alcohol, polyglycolic acid, poly(caprolactone / butylene succinate), poly(butylene succinate / adipate), poly(ethylene terephthalate / succinate), poly(tetramethylene adipate / terephthalate), polyhydroxybutyric acid, esterified starch, cellulose acetate, carboxymethylcellulose, carboxymethylcellulose salts, chitin, chitosan, collagen, fibronectin, alginic acid, alginate, starch, gelatin, and decellularizing materials. Collagen is preferred as the biodegradable polymer.

[0015] The methods for obtaining the above-mentioned decellularized material are not particularly limited, but examples include methods using surfactants, methods using enzymes, methods using oxidizing agents, methods using high hydrostatic pressure treatment, methods using freeze-thaw treatment, and methods using hypertonic electrolyte solutions.

[0016] In the tissue regeneration implant of this embodiment, the implant is preferably a sponge-like structure having a porous structure, and more preferably a collagen sponge. The diameter of the pore is preferably 1 μm or more and less than 50 μm, and more preferably 5 μm or more and 30 μm or less.

[0017] In the tissue regeneration implant of the present embodiment, the implant may be insolubilized with a chemical crosslinking agent. It is preferable that the physical strength is enhanced by the insolubilization treatment. As the strength, the tensile strength is 1N / mm 2 or more and 5N / mm 2 or less are exemplified.

[0018] In the tissue regeneration implant of the present embodiment, cells are not particularly limited, and may be cells collected from a living organism, may be cultured cells, or may be cells subjected to various genetic modification treatments such as knockdown or knock-in treatment of a specific gene. Examples of such cells include mesenchymal cells such as fibroblasts, synovial cells, chondrocytes and osteoblasts, or progenitor cells thereof; immune cells such as vascular endothelial cells, neutrophils, eosinophils, basophils, monocytes, T cells, B cells and dendritic cells; cardiomyocytes, epithelial cells, keratinocytes, nerve cells, hepatocytes, hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells. Among the above cells, chondrocytes are preferred.

[0019] The chondrocytes may be cells prepared by culturing primary cells isolated from animal cartilage tissue, or may be established cultured cells. Collection of animal cartilage tissue, recovery of chondrocytes from the cartilage tissue, and culture of the recovered chondrocytes can be performed by conventional methods. The number of passages of chondrocytes recovered from cartilage tissue is not particularly limited, but any of 13 passages or more, 12 passages or more, 11 passages or more, 10 passages or more, 9 passages or more, or 4 passages or more is preferred. Among them, 9 passages or more are more preferred, and 9 passages are even more preferred.

[0020] The mesenchymal stem cells described above are stem cells contained in bone marrow, adipose tissue, placenta, umbilical cord, dental pulp and the like. Mesenchymal stem cells can be prepared by culturing via a conventional method using primary cells isolated from animal bone marrow, adipose tissue, placenta, umbilical cord, dental pulp and the like.

[0021] The cells used in the tissue regeneration implant of this embodiment are preferably cells that are less susceptible to immune responses when transplanted into the body, more preferably cells isolated from infant-derived tissue, and even more preferably infant-derived chondrocytes. The above-mentioned infants are children aged 7 years or younger, 6 years or younger, 5 years or younger, 4 years or younger, 3 years or younger, or 2 years or younger, with children aged 2 years or younger being preferred. Another embodiment of the infant-derived chondrocytes is cells that highly express either or both of PD-L1 (Programmed death-ligand 1) and PD-L2 (Programmed death-ligand 2). In the present invention, high expression means that the expression is 3 times or more, 2 times or more, 1.5 times or more, 1.4 times or more, 1.3 times or more, 1.2 times or more, or 1.1 times or more higher than non-infant-derived chondrocytes such as adult-derived chondrocytes, and preferably 1.5 times or more. Another embodiment of the infant-derived chondrocytes is chondrocytes in which ossification is suppressed.

[0022] In this embodiment, cells in the tissue regeneration implant may be impregnated by immersing the implant in a suspension of cells in a solvent. The solvent is not particularly limited and can be appropriately determined considering the type of cells, etc. Examples of solvents in which the cells can survive include isotonic solutions, such as cell culture media, phosphate saline (PBS), physiological saline, lactated Ringer's solution, Ringer's solution, acetate Ringer's solution, and bicarbonate Ringer's solution. Among these, cell culture media, PBS, or a mixed solution of cell culture media and PBS is preferred. As for the cell culture media, it can be appropriately selected from known culture media considering the type of cells to be suspended, and can also be appropriately modified as needed. Specifically, the cell culture medium may be MEM, α-MEM, DMEM (Dulbecco's modified Eagle medium), IMDM, HamF10, HamF12, Medium199, RPMI1640, RITC80-7, MCDB104, MCDB105, MCDB153, MCDB201, MCDB202, Fisher's medium, or a mixture thereof. Furthermore, the cells in the tissue regeneration implant of this embodiment may be thawed cells that have been cryopreserved. The cryopreserved cells may be suspended in a solvent used for cryopreservation. Examples of solvents used for cryopreservation include an isotonic solution or cell culture medium to which 5.0 v / v% to 10.0 v / v% of dimethyl sulfoxide and 2.0 w / v% to 5.0 w / v% of albumin have been added.

[0023] The number of cells contained in the tissue regeneration implant of this embodiment is not particularly limited. The number of cells could be 5.0 × 10⁻⁶. 3 cells / mm 3 The above 3.0 × 10 4 cells / mm 3 The following are preferable. The lower limit of the number of cells in question is 3.0 × 10⁻⁶. 3 cells / mm 3 , 4.0×10 3 cells / mm 3 , 5.0×10 3 cells / mm 3 5.5 cells / mm 3 , 5.9×10 3 cells / mm 3 , 6.0×10 3 cells / mm 3 Preferably, it is one of the following: 3.0 × 10 3 cells / mm 3 More preferably, 5.9 × 10 3 cells / mm 3 That is even more preferable. From the viewpoint of preferring not to use too many cells, the upper limit for the number of cells is 2.0 × 10⁻⁶. 4 cells / mm 3 , 1.5×10 4 cells / mm 3 , 1.4×10 4 cells / mm 3 , 1.3 × 10 4 cells / mm 3 , 1.2 × 10 4 cells / mm 3 , 1.1 × 104 cells / mm 3 , 1.0 × 10 4 cells / mm 3 It is preferable that it be one of the following, 1.2 × 10 4 cells / mm 3 It is preferable that it be so. The upper and lower limits for the number of cells in question are 3.0 × 10⁶, based on the effects of inhibiting cell catabolism and degradation, and inhibiting cell enlargement. 3 cells / mm 3 Preferably, the above is true, and furthermore, from the viewpoint of further enhancing the early maturation of cells in the tissue regeneration implant and cell migration from the recipient, 3.0 × 10 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3 The following is preferable: 5.9 × 10 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3 The following are preferable.

[0024] The fibrin in the tissue regeneration implant of this embodiment may be fibrin produced by mixing and reacting fibrinogen and thrombin. Fibrinogen and thrombin may be in the form of dry powders, or they may be in the form of solutions dissolved in a suitable solvent. As a thrombin solution, for example, a solution of thrombin dissolved in purified water at a concentration of 100 units / mL to 500 units / mL can be used. As a fibrinogen solution, for example, a solution of fibrinogen dissolved in purified water at a concentration of 10 mg / mL to 200 mg / mL can be used. Both thrombin and fibrinogen may be purified products derived from animal blood, or synthetic products artificially synthesized using a microbial expression system or the like may be used.

[0025] Factor XIII and calcium ions are coagulation factors that strengthen the fibrin network structure. Furthermore, while fibrin gel in the transplant cell composition is degraded by plasmin in the recipient tissue, aprotinin inhibits plasmin. Therefore, these may be dissolved in either a thrombin solution or a fibrinogen solution, respectively. For example, the thrombin solution may contain calcium chloride, and the fibrinogen solution may contain factor XIII and aprotinin.

[0026] The fibrin may be fibrin injected into the implant after the cell-supported implant has been transplanted into the body. Alternatively, the fibrin may be fibrin produced by injecting a fibrinogen solution and a thrombin solution into a cell-supported implant and allowing them to react within the implant.

[0027] <Manufacturing method for tissue regeneration implants> The method for manufacturing a tissue regeneration implant according to this embodiment (hereinafter also simply referred to as "the manufacturing method according to this embodiment") includes a cell impregnation step of impregnating an implant with cells, and a fibrin impregnation step of impregnating the implant carrying the cells with fibrin after the cell impregnation step.

[0028] The cell impregnation step in the manufacturing method of this embodiment is not particularly limited as long as it can impregnate the implant with cells. Specifically, one method is to impregnate the implant with cells while the cells are suspended in a solvent, thereby supporting the cells on the implant. The tissue regeneration implant, cells, and solvent used in the cell impregnation process are the same as those described above under <Tissue Regeneration Implant>.

[0029] In the cell impregnation process, 3.0 × 10⁻⁶ cells are applied to the implant. 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3It is preferable to support the following cells.

[0030] The fibrin impregnation step in the manufacturing method of this embodiment is not particularly limited as long as it can impregnate the implant with fibrin after the cell impregnation step. Specifically, one method is to impregnate the cell-supported implant with fibrin obtained by reacting fibrinogen and thrombin, thereby supporting fibrin on the implant. Alternatively, one method is to inject fibrinogen and thrombin into the implant and allow them to react within the implant to support fibrin on the implant. The tissue regeneration implant, cells, fibrin, fibrinogen, thrombin, and solvent used in the fibrin impregnation process are the same as those described above in the section on <tissue regeneration implant>.

[0031] In the fibrin impregnation process, it is preferable to inject a fibrinogen solution and a thrombin solution into the implant and allow them to react within the implant to produce fibrin, and it is more preferable to inject the thrombin solution into the implant first, and then inject the fibrinogen solution into the implant and allow them to react within the implant to produce fibrin.

[0032] In the manufacturing method of this embodiment, an implantation step may be included in which an implant carrying cells is transplanted into a living body after the cell impregnation step and before the fibrin impregnation step.

[0033] <Other Embodiments> In one embodiment, the present invention provides a treatment method comprising implanting a therapeutically effective amount of a tissue regeneration implant into a living body, wherein the tissue regeneration implant comprises an implant, cells supported on the implant, and fibrin supported on the implant.

[0034] In one embodiment, the present invention provides the use of an implant, cells supported on the implant, and fibrin supported on the implant in the manufacture of a tissue regeneration implant. [Examples]

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] [Test Example 1] The expression of type I collagen and ACAN (aggrecan) in collagen sponges carrying both cells and fibrin (hereinafter also simply referred to as "cell and fibrin-carrying collagen sponges") was evaluated using infant-derived chondrocytes.

[0037] ≪1-1: Human infant-derived chondrocytes≫ Chondrocytes (6 passages) established from cartilage tissue of infant donors were cultured for 2 passages in a flask in DMEM containing 10 v / v% FBS. Next, the infant-derived chondrocytes were detached with trypsin and centrifuged. The supernatant was then removed. Next, 3 × 10⁻⁶ 7 The cells were suspended in 1.2 mL of cryopreservation solution at a concentration of cells / mL. This cell suspension was then packed into cryotubes (Nipro Corporation) and stored at -80°C for at least one day. Afterward, the cells were moved to a liquid nitrogen vapor phase for storage.

[0038] The above cryopreservation solution was prepared using the following method. Specifically, a solution was prepared by mixing 7.5 v / v% DMSO (Nipro CryoServe NDM-50) and DMEM (ThermoFisher DMEM 12100), to which 2.5 w / v% albumin (Wuhan Healthgen Biotechnology HYC002C02) was added to create a cryopreservation solution.

[0039] ≪1-2: Method for preparing a collagen sponge that supports cells≫ After removing the cryotubes stored in step ≪1-1≫ above, the cell suspension was thawed by heating at 37°C. After thawing, 23 μL of cell suspension (6.9 × 10⁶ cells) was used. 5 Cells were dropped onto a collagen sponge Mighty (5mm in diameter x 3mm in height) (CSM-25, manufactured by KOKEN Co., Ltd.) to create a collagen sponge that supports cells (hereinafter also simply referred to as "cell-supporting collagen sponge").

[0040] ≪1-3: Method for preparing cell and fibrin-supported collagen sponges≫ First, 1 mL of DMEM was added to 1 mL of Volheal fibrinogen (80 mg: KM Biologics) to prepare a fibrinogen-DMEM solution (hereinafter also simply referred to as "fibrinogen solution"). Next, 0.75 mL of DMEM was added to 1 mL of Volheal thrombin (250 units: KM Biologics) to prepare a thrombin-DMEM solution (hereinafter also simply referred to as "thrombin solution").

[0041] After removing the cryotubes stored in step ≪1-1≫ above, the cell suspension was thawed by heating at 37°C. After thawing, 23 μL of cell suspension (6.9 × 10⁶ cells) was used. 5 Cells, 2.5 μL of fibrinogen solution, and 3 μL of thrombin solution were added in that order to a collagen sponge Mighty (5 mm in diameter x 3 mm in height) (CSM-25, manufactured by KOKEN Co., Ltd.) to prepare cell- and fibrin-carrying collagen sponges.

[0042] ≪1-5: Evaluation of cells and fibrin-supported collagen sponges (evaluation of type I collagen and ACAN expression)≫ Cell-supported collagen sponges and cell- and fibrin-supported collagen sponges were seeded in 96-well plates, and 300 μL of induction medium (DMEM supplemented with 10 v / v% FBS, 400 μg / mL BMP-7, 200 ng / mL IGF-1, and 10 μg / mL insulin) was added to each well. The cells were then cultured at 37°C under an 8% CO2 atmosphere for 1, 2, or 4 weeks. RNA was extracted from the cultured cells and PCR was performed according to standard procedures. The mRNA expression levels of type I collagen and ACAN (aggrecan) were analyzed by ratio to the mRNA level of the internal standard GAPDH.

[0043] As shown in Figures 1A and 1B, the expression levels of type I collagen (Col1 in the figures) and ACAN were high in both cells and fibrin-supported collagen sponges immediately after the start of culture. Therefore, it was revealed that cells and fibrin-supported collagen sponges exhibited functions closer to those of living tissues at an earlier stage compared to cell-supported collagen sponges.

[0044] [Test Example 2] The evaluation of cells and fibrin-loaded collagen sponges was carried out by reducing the concentration of cells loaded onto the collagen sponge to half that of Test Example 1.

[0045] ≪2-1: Method for preparing cell and fibrin-supported collagen sponges≫ First, a thrombin solution was prepared in the same manner as described in Test Example 1. Next, after removing the cryotubes stored in step ≪1-1≫ above, the cell suspension was thawed by heating at 37°C. After thawing, the cell suspension was diluted 2-fold with DMEM. Then, 23 μL of the 2-fold diluted cell suspension (3.45 × 10⁶ cells) was added. 5 Cells, 2.5 μL of fibrinogen solution, and 3 μL of thrombin solution were added in that order to a collagen sponge Mighty (5 mm in diameter x 3 mm in height) (CSM-25, manufactured by KOKEN Co., Ltd.) to prepare cells and fibrin-carrying collagen sponges (hereinafter also simply referred to as the "low concentration group (L)"). Furthermore, for comparison with the low-concentration group (L), cells and fibrin-supported collagen sponges were prepared in the same manner as described in Test Example 1 (hereinafter also simply referred to as the "standard group (S)").

[0046] ≪2-2: Evaluation of Cells and Fibrin-Loaded Collagen Sponges (Evaluation of Type I Collagen and ACAN Expression)≫ The expression of type I collagen and ACAN was evaluated in the low-concentration group (L) or the standard group (S) in the same manner as described in Test Example 1.

[0047] As shown in Figures 2A and 2B, no difference was observed in the expression levels of Col1 or ACAN between the low-concentration group (L) and the standard group (S). Therefore, 3.5 × 10⁶ for a collagen sponge (diameter 5 mm × height 3 mm) 5 cells or larger: 6.9 × 10 5 Cells smaller than 10 cells (cell concentration: 5.9 × 10⁻⁶) 3 cells / mm 3 The above 1.2 × 10 4 cells / mm 3 It was revealed that collagen sponges carrying fibrin (as described below) exhibit functions closer to those of biological tissue at an earlier stage.

[0048] [Test Example 3] We evaluated the fusion of cells and fibrin-loaded collagen sponges with recipient tissues after transplantation into vivo.

[0049] ≪3-1: Method for creating a porcine meniscus with embedded cell-supported collagen sponge≫ Cell-supported collagen sponges were prepared in the same manner as described in Test Example 1. Next, a 4 mm diameter hole was made in the meniscus taken from a pig's knee, and a cell-supported collagen sponge was embedded. The meniscus with the embedded collagen sponge was transferred to a 6-well plate and cultured in DMEM supplemented with 10 v / v% FBS at 37°C under an 8% CO2 atmosphere for 2 or 4 weeks.

[0050] ≪3-2: Method for preparing porcine meniscus with embedded cell and fibrin-supported collagen sponge≫ A thrombin solution was prepared in the same manner as described in Test Example 1. Next, thrombin solution was dropped onto the area in the porcine meniscus prepared by the method described in <3-1> above where the collagen sponge was embedded. Furthermore, fibrinogen solution was dropped onto the area. The meniscus was then transferred to a 6-well plate and cultured in DMEM with 10 v / v% FBS added at 37°C under an 8% CO2 atmosphere for 2 or 4 weeks.

[0051] ≪3-3: Evaluation of Cells and Fibrin-Loaded Collagen Sponge (Evaluation of Pig Cell Migration Ability)≫ Fixed sections were prepared from the boundary region between the pig meniscus and the collagen sponge. Then, using the pig chromosome 1 FISH probe Cy3-labeled (P10-20, Chromosome Science Lab Co., Ltd.) and the human XY chromosome FISH probe FITC-labeled (HXY-10, Chromosome Science Lab Co., Ltd.), porcine cells and human infant-derived chondrocytes were stained by FISH (fluorescence in situ hybridization) according to standard procedures, and the number of porcine cells migrating to the collagen sponge was evaluated. DAPI staining was also performed as a control.

[0052] As shown in Figures 3 and 4, cell and fibrin-carrying collagen sponges (cultured for 2 weeks: Figure 3 bottom panel, cultured for 4 weeks: Figure 4 bottom panel) showed more migration of porcine cells toward the collagen sponge compared to cell-carrying collagen sponges (cultured for 2 weeks: Figure 3 top panel, cultured for 4 weeks: Figure 4 top panel). Therefore, it is clear that cell and fibrin-carrying collagen sponges are more readily integrated with the recipient tissue than cell-carrying collagen sponges.

[0053] [Test Example 4] We evaluated the immune responses of cells and cells used in fibrin-loaded collagen sponges. ≪4-1:Adult-derived chondrocytes≫ Except for using chondrocytes (6 passages) established from the cartilage tissue of an adult donor, a collagen sponge carrying adult-derived chondrocytes was prepared in the same manner as described in Test Example 1.

[0054] ≪4-2: Assessment of Immune Response≫ Collagen sponges carrying human infant-derived chondrocytes (the same cells as described in Test Example 1) or adult-derived chondrocytes were seeded in 96-well plates, and 200 μL of complete medium (10 v / v% FBS, high glucose DMEM supplemented with 1% penicillin-streptomycin) was added to each well. The cells were then cultured at 37°C in a 5% CO2 environment for 3 days. RNA was extracted from the cultured cells and PCR was performed according to standard procedures. The mRNA expression levels of PD-L1 (Programmed cell Death ligand 1) and PD-L2 (Programmed cell Death ligand 2) were analyzed by ratio to the mRNA level of the internal standard GAPDH.

[0055] As shown in Figures 5A and 5B, infant-derived chondrocytes showed higher levels of PD-L1 and PD-L2 expression and were less susceptible to immune responses compared to adult-derived chondrocytes.

[0056] [Test Example 5] The evaluation of the cell-supported collagen sponge was carried out by using a cell concentration that was one-quarter or half of that used in Test Example 2.

[0057] ≪6-1: Method for preparing cell-supported collagen sponges≫ The human infant-derived cartilage cells described in Test Example 1 were subjected to a 0.9 × 10⁶ measurement. 5 cells (LD group), 1.8×10 5 cells (MD group), 3.6×10 5 Cells (HD group) were impregnated into Mighty (5mm diameter x 3mm height) (CSM-25, manufactured by KOKEN), and cell-supported collagen sponges (each 1.5 x 10 3 cells / mm 3 , 3.0×103 cells / mm 3 , 6.0×10 3 cells / mm 3 These were prepared. These were cultured for 15 days at 37°C in a 5% CO2 atmosphere in DMEM supplemented with 10 ng / mL TGF-β3, 50 μg / mL L-ascorbate-2-phosphate, 40 μg / mL L-proline, 0.5% penicillin-streptomycin, and 1% ITS+PREMIX.

[0058] ≪6-2: Evaluation of cell-supported collagen sponges (evaluation of expression of type I collagen, type II collagen, MMP3, MMP13, RUNX2, and type X collagen)≫ Expression levels of type I collagen, type II collagen, MMP3, MMP13, RUNX2, and type X collagen were evaluated in the LD, MD, and HD groups in the same manner as described in Test Example 1.

[0059] As shown in Figure 6, the MD and HD groups showed a tendency for the expression of catabolic genes (MMP13) and hypertrophic genes (type X collagen) to be suppressed.

[0060] [Test Example 6] Gene expression in cells used in cell-supported collagen sponges was evaluated.

[0061] ≪7-1:Adult-derived chondrocytes≫ Except for using chondrocytes (passages 3 or 4) established from cartilage tissue of adult donors and adult-derived synovial cells (passages 3 or 4) established from synovial tissue, collagen sponges carrying adult-derived chondrocytes or adult-derived synovial cells were prepared in the same manner as described in Test Example 6.

[0062] ≪7-2: Gene Expression Evaluation≫ The mRNA expression levels of collagen sponges carrying human infant-derived chondrocytes (the same cells as described in Test Example 1), adult-derived chondrocytes, or adult-derived synovial cells were analyzed by RNA-seq according to standard procedures.

[0063] The analysis results are shown in Figure 7. The left panel shows 500 genes with high mRNA expression levels in collagen sponges supported by infant-derived chondrocytes (PDCs) and adult-derived chondrocytes (CDCs) or adult-derived synovial cells (SDCs). The right panel of Figure 7 shows metabolic pathways that contain many of the genes whose expression levels were reduced in infant-derived chondrocytes (the gene groups enclosed in squares in the left panel). As shown in Figure 7, infant-derived chondrocytes were found to have properties that facilitate meniscus regeneration, such as being more likely to suppress ossification, compared to adult-derived chondrocytes or adult-derived synovial cells. [Industrial applicability]

[0064] According to the present invention, it is possible to provide a tissue regeneration implant that exhibits good tissue regeneration and implant replacement in the recipient. For this reason, the present invention is particularly useful in the field of regenerative medicine.

Claims

1. A tissue regeneration implant that is transplanted into the living body, A tissue regeneration implant comprising an implant, cells supported on the implant, and fibrin supported on the implant.

2. The tissue regeneration implant according to claim 1, wherein the implant is a collagen sponge.

3. A tissue regeneration implant according to claim 1 or 2, which is implanted within the meniscus.

4. The tissue regeneration implant according to claim 1 or 2, wherein the fibrin is fibrin injected into the implant after the implant carrying the cells has been transplanted into the living body.

5. The tissue regeneration implant according to claim 1 or 2, wherein the fibrin is fibrin produced by injecting a fibrinogen solution and a thrombin solution into the implant carrying the cells and allowing them to react within the implant.

6. The tissue regeneration implant according to claim 1 or 2, wherein the cells are cartilage cells.

7. The tissue regeneration implant according to claim 6, wherein the chondrocytes are infant-derived chondrocytes.

8. For the aforementioned implant, 3.0 × 10 ３ cells / mm ３ The above 1.2 x 10 ４ cells / mm ３ The tissue regeneration implant according to claim 1 or 2, comprising the following cells.

9. A method for manufacturing tissue regeneration implants to be implanted in a living organism, The cell impregnation process involves impregnating the implant with cells, Following the cell impregnation step, a fibrin impregnation step is performed in which fibrin is impregnated into the implant carrying the cells. A method for manufacturing tissue regeneration implants, including the method described above.

10. The method for manufacturing a tissue regeneration implant according to claim 9, wherein the implant is a collagen sponge.

11. A method for manufacturing a tissue regeneration implant according to claim 9 or 10, comprising a transplantation step of transplanting the implant carrying the cells into the living body after the cell impregnation step and before the fibrin impregnation step.

12. The method for manufacturing a tissue regeneration implant according to claim 9 or 10, wherein the biological tissue is located within the meniscus.

13. A method for producing a tissue regeneration implant according to claim 9 or 10, wherein in the fibrin impregnation step, a fibrinogen solution and a thrombin solution are injected into the implant impregnated with cells and reacted within the implant to generate fibrin.

14. The method for producing a tissue regeneration implant according to claim 9 or 10, wherein the cells are cartilage cells.

15. The method for producing a tissue regeneration implant according to claim 14, wherein the cartilage cells are infant-derived cartilage cells.

16. In the cell impregnation step, 3.0 × 10 ３ cells / mm ３ The above 1.2 x 10 ４ cells / mm ３ A method for manufacturing a tissue regeneration implant according to claim 9 or 10, wherein the implant is supported by the following cells.

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