Graft material for tendon or ligament transplants

Abstract

The present invention provides a transplantation material for reconstruction of tendons or ligaments. According to the present invention, provided is a transplantation material that is for reconstruction of tendons or ligaments and that include a cell sheet containing adipose-derived stem cells.

Description

[Technical field]

[0001] The present invention relates to a graft material for tendon or ligament transplants. [Background technology]

[0002] Anterior cruciate ligament reconstruction (ACL reconstruction) is a surgical procedure to replace a torn anterior cruciate ligament with a graft, thereby restoring the function of the damaged ligament. Torn ligaments rarely heal naturally, so ACL reconstruction is essential for athletes to return to sports. In ACL reconstruction, an autologous tendon graft is used, and many top-level athletes in soccer, rugby, American football, basketball, etc. are able to return to play after surgery and perform as well as they did before their injury.

[0003] To date, various treatments have been used for ACL reconstruction, but treatment using artificial ligaments has poor long-term outcomes, making the development of new treatments a challenge. 11,31 Recently, there have been reports of the application of various stem cells to existing ACL reconstruction surgery. 10 , Reporting using MSC 44 , a report using tendon-derived stem cells (Tenocytes) 30 , reported using ADSCs 21 Various types of cells are directly injected into the joint. 32 , or by mixing it with fibrin glue and injecting it into the joint. 16 There have been reports of plants being transplanted in sheets. 18 While some papers report technical significance, others report that no technical significance was obtained. 1,37 In addition, ADSCs show a tendency to differentiate into osteochondrocytes depending on the environment. 2,24,27,51 However, there are also reports that they show a tendency to differentiate into tendon cells. 22,26 It has also been reported that heterotopic bone formation may occur as a side effect. 8 Kosaka's and Zhang's applied ADSC suspension to ACL reconstruction, and both reported good results, although the ADSC transplantation method was injection.21,50 . Summary of the Invention

[0004] The present invention provides a transplant material for tendon or ligament transplantation. The present invention also provides a transplant material comprising a cell sheet comprising ADSCs.

[0005] The present inventors have found that in an animal model in which an autologous tendon wrapped with an ADSC sheet was transplanted by ACL reconstruction surgery, ADSCs survive inside the graft and in the surrounding tissue, increase factors that promote tissue regeneration, improve the number, thickness, and density of collagen fibers in the graft, promote calcification of the bone in contact with the ADSCs in the graft, inhibit bone tunnel enlargement after graft transplantation, and increase the maximum breaking load and rigidity of the graft. The present invention is based on these findings.

[0006] According to the present invention, the following inventions can be provided. [1] A graft material for tendon or ligament reconstruction comprising a cell sheet containing adipose-derived stem cells. [2] The transplant material described in [1] above, comprising the layered cell sheets. [3] The transplant material described in [1] or [2] above, wherein the cell sheet maintains an adhesive protein on the surface of the sheet. [4] The transplant material according to any one of [1] to [3] above, which is used by being attached to the surface of a graft for tendon or ligament reconstruction. [5] The transplant material according to any one of the above [1] to [3], which is used by being attached to the surface of a junction between a tendon or ligament and bone in a graft for tendon or ligament reconstruction. [6] The transplant material according to any one of the above [1] to [3], which is used by being attached to a surface other than the junction between the tendon or ligament and the bone of a graft for tendon or ligament reconstruction. [7] A transplant material comprising a cell sheet containing adipose-derived stem cells, for use in inhibiting bone tunnel enlargement at a graft junction after tendon or ligament reconstruction surgery, or the transplant material described in any of [1] to [6] above. [8] The transplant material according to any one of the above [1] to [7], wherein the cell sheet contains 90% or more cells positive for CD29, CD44, CD73, CD90, CD105, and CD166, and 5% or less cells positive for CD14, CD31, CD45, and Lin1. [9] A product comprising a container containing the transplant material described in any one of [1] to [8] above.

[10] A combination of a graft for tendon or ligament reconstruction and the transplant material described in any one of [1] to [8] above attached to its surface.

[11] The combination according to

[10] above, wherein the surface is a surface of a junction between a tendon or ligament of a graft and bone.

[12] The combination according to

[10] above, wherein the surface is a surface other than the junction of the tendon or ligament of the graft with the bone.

[13] A transplant material comprising the cell sheet defined in any one of [1] to [8] above, which is transplanted in a state attached to a transplant graft, thereby promoting the production of type 1 collagen in the graft.

[14] A transplant material comprising the cell sheet defined in any one of [1] to [8] above, which is transplanted in a state attached to a transplant graft, thereby suppressing the production of type III collagen in the graft.

[15] A transplant material comprising the cell sheet defined in any one of [1] to [8] above, which is transplanted in a state attached to a transplant graft, thereby increasing the collagen fiber diameter in the graft.

[16] A transplant material comprising the cell sheet defined in any one of [1] to [8] above, which is transplanted in a state attached to a transplant graft, thereby improving the occupancy rate of collagen fibers in the cross section of collagen fibers in the graft.

[17] A transplant material comprising the cell sheet defined in any one of [1] to [8] above, which is transplanted in a state attached to a transplant graft, thereby for use in suppressing rupture of the graft after transplantation.

[18] The transplant material according to any one of [1] to [8] above, further comprising a transplant graft.

[19] A method of reconstructing a tendon or ligament in a subject in need thereof, comprising: (i) fixing a graft to a site in need of tendon or ligament reconstruction in a subject, wherein the fixed graft has a cell sheet comprising adipose-derived stem cells on a surface thereof, or (ii) fixing the graft to a site in the subject where tendon or ligament needs to be reconstructed; and attaching a cell sheet containing adipose-derived stem cells to the surface of the fixed graft. method.

[20] A method of reconstructing a tendon or ligament in a subject in need thereof, comprising: (i) fixing a graft to a site in a subject requiring tendon or ligament reconstruction, wherein the fixed graft has a cell sheet on its surface comprising adipose-derived stem cells.

[21] A method of strengthening a reconstructed tendon or ligament in a subject, comprising: The method includes attaching a cell sheet containing adipose-derived stem cells to a surface of a graft fixed to a site that needs to be reconstructed. method.

[0007] In ACL reconstruction surgery, there are cases where the graft is torn after transplantation, and in such cases, reoperation is required. The transplant material of the present invention can reduce the need for reoperation. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows the placement of the semitendinosus tendon (ST) in anterior cruciate ligament reconstruction. The ST is covered in the center with a cell sheet. The ST is also covered on the interface with the bone with a cell sheet. [Diagram 2] Figure 2 shows the changes in rabbit weight, graft length, and graft diameter over time after transplantation. "w" in the graph indicates the number of weeks since surgery. [Diagram 3] Figure 3 shows the results of histological evaluation of the grafts after implantation. "B" represents bone, "G" represents graft, and arrowheads indicate Sharpey's fibers, chondrocytes, and lamellar bone. [Figure 4] FIG. 4 shows the progress of the score after tendon-bone healing. [Diagram 5] Figure 5 shows fluorescent staining images showing the migration and engraftment of transplanted adipose-derived stem cells (ADSCs) into the tissue surrounding the graft. ADSCs were fluorescently stained before transplantation. [Figure 6] Figure 6 shows that transplanted adipose-derived stem cells (ADSCs) migrated and engrafted inside the graft after transplantation. ADSCs were fluorescently stained before transplantation. [Figure 7] FIG. 7 shows the time-dependent change in the area of ​​the bone tunnel created during graft implantation. [Figure 8] Figure 8 shows the time course of bone mineral content (volume ratio) relative to total tissue after graft implantation. The left panel of Figure 8 shows the volume ratio when the area with a calcification level of 400 mg / ml or more is considered to be bone, and the right panel of Figure 8 shows the volume ratio when the area with a calcification level of 800 mg / ml or more is considered to be bone. [Figure 9] FIG. 9 shows an electron microscope image of a cross section of the graft after implantation (Panel A), as well as the number (Panel B), thickness (Panel C), and thickness distribution (Panel E) of collagen fibers, and fiber density (Panel E). [Figure 10] The upper panel of Fig. 10 is a photograph showing the state of the biomechanical experiment. Panel A of Fig. 10 shows the maximum breaking load (N) of the graft after implantation. Panel B of Fig. 10 shows the stiffness value (N / mm) of the graft after implantation. [Figure 11] Figure 11 shows the patterns of fracture: the left panel shows the graft coming out of the tibia, the center panel shows the graft breaking in the middle, and the right panel shows the graft coming out of the femur. [Figure 12] FIG. 12 shows the relationship between the bone mineral content (volume ratio) relative to the total tissue after graft implantation and the maximum breaking load. [Figure 13] FIG. 13 shows the mRNA expression of various genes in the grafts. [Figure 14] FIG. 14 shows the mRNA expression of various genes in the grafts. Detailed Description of the Invention

[0009] As used herein, a "subject" refers to a vertebrate, such as a bird or a mammal, for example, a mammal, such as a mouse, a rat, a hamster, a guinea pig, a horse, a cow, a pig, a goat, a sheep, a donkey, a dog, and a cat, as well as a primate, such as a monkey, a chimpanzee, a gorilla, an orangutan, a bonobo, or a human, particularly a human. As used herein, a subject is used in the sense of including a human, as described above, and the term "non-human" is used to exclude a human. As used herein, a "subject" refers to a subject to which a graft is transplanted.

[0010] As used herein, "treatment" refers to medical intervention in a subject. Treatment is used in a sense that includes therapeutic treatment. Therapeutic treatment can cause a therapeutic effect such as improvement, inhibition of deterioration, reduction in the rate of deterioration, cessation, and cure of a disease, disorder, or condition. As used herein, a "therapeutically effective amount" is an amount that produces a therapeutic effect.

[0011] As used herein, the term "transplant material" refers to a biomaterial used for transplantation into the body of a subject. The biomaterial may be an artificial material, a natural material, or a processed product of a natural material. The transplant material is preferably biocompatible. The transplant material may be autologous or allogeneic. The cell sheet containing the adipose-derived stem cells may be autologous or allogeneic to the subject to be transplanted. The term transplant material does not necessarily require the presence of a graft. However, the transplant material may include a graft. That is, the transplant material may include a cell sheet or may further include a graft in addition to the cell sheet.

[0012] As used herein, "adipose-derived stem cells" (ADSCs) are cells isolated from adipose tissue and can differentiate into multiple mature cells, including adipocytes, osteoblasts, and chondrocytes. Adipose-derived stem cells can be human adipose-derived stem cells. Adipose-derived stem cells can express one or more or all selected from the group consisting of CD29, CD44, CD73, CD90, CD105, and CD166 (or can be positive for one or more or all). Adipose-derived stem cells can not express one or more or all selected from the group consisting of CD14, CD31, CD45, and Lin1 (or can be negative for one or more or all).

[0013] In this specification, a "cell sheet" refers to a sheet-shaped structure consisting of a single layer of cells. Cells are usually cultured in the form of a single layer sheet on a solid surface. When intercellular adhesion is destroyed, the cells dissociate to the single cell level, but a cell sheet can be obtained by recovering the cells without destroying (or not destroying completely) the intercellular adhesion. The intercellular adhesion is destroyed by treatment with a protease such as trypsin. In order to recover a cell sheet without destroying the intercellular adhesion, a temperature-responsive culture dish can be used. Multiple cell sheets can be used by stacking (i.e., layering).

[0014] As used herein, "extracellular matrix" refers to an insoluble substance present outside cells in vivo or in vitro. The extracellular matrix may contain adhesive proteins involved in cell adhesion. The adhesive proteins can be secreted by cells to the cell surface during culture. In cell sheets, the adhesive proteins can be maintained on the sheet surface. This allows the cell sheet to maintain its adhesiveness to other cells. Cell sheets that maintain their adhesiveness have increased adhesion to tissues and organs, and may be useful for transplantation purposes.

[0015] As used herein, a "graft" refers to a tissue or a portion thereof that is transplanted. A graft is also called a transplant. For example, in anterior cruciate ligament reconstruction, tendon or ligament grafts such as hamstring tendon (including semitendinosus tendon), patellar tendon, and iliotibial band can be used. In a preferred embodiment, the graft is an autologous graft to the subject. In a preferred embodiment, the graft is an allogeneic graft to the subject.

[0016] According to the present invention, there is provided a cell sheet comprising adipose-derived stem cells. According to the present invention, there is provided a transplant material comprising a cell sheet comprising adipose-derived stem cells. According to the present invention, there is provided a transplant material for tendon or ligament reconstruction comprising a cell sheet comprising adipose-derived stem cells.

[0017] The cell sheet containing the adipose-derived stem cells of the present invention contains, for example, 80% or more, 85% or more, 90% or more, or 95% or more of cells positive for CD29, CD44, CD73, CD90, CD105, and CD166. The cell sheet containing the adipose-derived stem cells of the present invention contains, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of cells positive for any of CD14, CD31, CD45, and Lin1. The cell sheet containing the adipose-derived stem cells of the present invention contains, for example, 80% or more, 85% or more, 90% or more, or 95% or more cells positive for one or more selected from the group consisting of CD29, CD44, CD73, CD90, CD105, and CD166, and / or 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less cells positive for one or more selected from the group consisting of CD14, CD31, CD45, and Lin1. The cell sheet containing the adipose-derived stem cells of the present invention contains, for example, 90% or more cells positive for CD29, CD44, CD73, CD90, CD105, and CD166, and 5% or less cells positive for any of CD14, CD31, CD45, and Lin1. The cell sheet containing the adipose-derived stem cells of the present invention contains, for example, 95% or more cells positive for CD29, CD44, CD73, CD90, CD105, and CD166, and 2% or less cells positive for any of CD14, CD31, CD45, and Lin1. In one embodiment, the adipose-derived stem cells may be positive for CD29, CD44, CD73, CD90, CD105, and CD166, and may be negative for any of CD14, CD31, CD45, and Lin1. Here, the percentage is calculated based on the number of cells. Whether each factor is positive or negative can be determined by flow cytometry using an antibody linked to a fluorescent dye. By comparing with either or both of the positive control cells and the negative control cells, the signal intensity in the case of negative and positive can be grasped. By analyzing the cells by flow cytometry, whether they are positive or negative can be determined by whether they are negative or show strong fluorescence that is significantly different from negative.

[0018] The cell sheet containing the adipose-derived stem cells of the present invention can be used by attaching it in a sheet-like form to the surface of a transplant graft. The cell sheet containing the adipose-derived stem cells of the present invention can be used by attaching it in a sheet-like form to the surface of a tendon graft or a ligament graft. Therefore, the cell sheet containing the adipose-derived stem cells of the present invention can be a transplant material used for the reconstruction of tendons or ligaments. It is not necessary to use an adhesive (e.g., fibrin glue) to attach the cell sheet to the surface of a transplant graft.

[0019] The cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of a tendon or ligament graft, which prevents the enlargement of the bone hole in contact with the graft and improves bone density. In this way, the cell sheet containing the adipose-derived stem cells of the present invention can be attached to the vicinity of the bone hole in contact with the graft to prevent the enlargement of the bone hole after graft transplantation and induce an improvement in bone density. In addition, it can promote the formation of bone with a lamellar structure at the tendon-bone junction. Therefore, the cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of the graft that will be the junction with the bone. In one embodiment, in order to prevent the enlargement of the bone hole after graft transplantation or induce an improvement in bone density, the cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of the graft that will be the junction with the bone. The cell sheet containing the adipose-derived stem cells of the present invention can be attached to the graft surface, preferably in the form of a sheet.

[0020] The cell sheet containing the adipose-derived stem cells of the present invention can be attached in a sheet form to the surface of a tendon or ligament graft, which can provide the following effects: (1) promoting the production of type 1 collagen, (2) inhibiting the production of type 3 collagen, (3) increasing the collagen fiber diameter (e.g., average diameter), and (4) improving the occupancy rate of collagen fibers in the collagen fiber cross section. Therefore, the cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of the graft other than the junction with the bone. In one embodiment, the cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of the graft other than the junction with the bone for use in one or more applications selected from the group consisting of (1) promoting the production of type 1 collagen, (2) inhibiting the production of type 3 collagen, (3) increasing the collagen fiber diameter (e.g., average diameter), and (4) improving the occupancy rate of collagen fibers in the collagen fiber cross section. Therefore, according to the present invention, the cell sheet containing the adipose-derived stem cells of the present invention or a transplant material containing the cell sheet can be used to (1) promote the production of type 1 collagen in a graft. Also according to the present invention, the cell sheet containing the adipose-derived stem cells of the present invention or a transplant material containing the cell sheet can be used in a graft to (2) suppress type III collagen production. Also according to the present invention, the cell sheet containing the adipose-derived stem cells of the present invention or a transplant material containing the cell sheet can be used in a graft to (3) increase collagen fiber diameter (e.g., average diameter). Also according to the present invention, the cell sheet containing the adipose-derived stem cells of the present invention or a transplant material containing the cell sheet can be used in a graft to (4) increase the occupancy rate of collagen fibers in the collagen fiber cross section. The cell sheet containing the adipose-derived stem cells of the present invention can be attached to the graft surface, preferably in the form of a sheet.

[0021] The cell sheet containing the adipose-derived stem cells of the present invention can be used to suppress detachment from the tibia and femur after graft transplantation, and rupture of the graft after transplantation. The cell sheet containing the adipose-derived stem cells of the present invention can be preferably used to suppress rupture of the graft after transplantation. As a result, the cell sheet containing the adipose-derived stem cells of the present invention can reduce the need for reoperation after graft transplantation (e.g., after ACL reconstruction surgery).

[0022] The cell sheet containing the adipose-derived stem cells of the present invention may be attached in a layered manner. In this manner, it is believed that the physiological action of the cell sheet is enhanced. The layering may be performed by layering two or more layers of sheets. The layering may be performed by layering 2 to 10 layers, 2 to 5 layers, or 2 or 3 layers of sheets.

[0023] The cell sheet containing the adipose-derived stem cells of the present invention can be used by attaching it to the surface of tendon or ligament graft in the form of a sheet, and the attachment can be performed so as to cover the graft surface. The attachment can also be performed so as to wrap the sheet around the graft surface. The attachment can also be performed so as to wrap the layered sheet around the graft surface. The layering can be two to several layers, for example, three, four, five, six or more layers.

[0024] In one aspect of the present invention, the cell sheet comprising the adipose-derived stem cells of the present invention can be provided in a sheet-like state attached to the surface of a tendon or ligament graft. Alternatively, the cell sheet comprising the adipose-derived stem cells of the present invention can be provided in combination with a tendon or ligament graft. In one aspect, the cell sheet comprising the adipose-derived stem cells of the present invention is attached to the surface of the tendon or ligament graft at the junction with the bone. In one aspect, the cell sheet comprising the adipose-derived stem cells of the present invention is attached to the surface of the tendon or ligament graft other than the junction with the bone.

[0025] Adipose-derived stem cells can be isolated from aspirated fat tissue. Separation and purification of adipose-derived stem cells from adipose tissue can be performed with reference to known methods previously reported. For example, the method described in the literature Arterioscler Thromb Vasc Biol. 29:1723-1729, 2009 can be used. Alternatively, commercially available human adipose-derived stem cells can be used. Adipose-derived stem cells can be human cells. Adipose-derived stem cells can be autologous cells. Adipose-derived stem cells can be allogeneic cells. Human adipose-derived stem cells can be cells established for medical use and quality-controlled (e.g., autologous cells, particularly allogeneic cells).

[0026] Adipose-derived stem cells can be cultured by methods well known to those skilled in the art. The culture can be adhesion culture. The cell sheet can be prepared by various methods well known to those skilled in the art. For example, the cell sheet can be prepared using a temperature-responsive culture dish. The culture surface of the temperature-responsive culture dish is coated with a temperature-responsive polymer. The temperature-responsive polymer is hydrophobic at normal culture temperatures (e.g., about 37°C), but when the temperature is lowered, it changes to hydrophilic in a temperature-dependent manner. The temperature at which the polymer changes from hydrophobic to hydrophilic, or from hydrophilic to hydrophobic, is called the lower critical solution temperature (LCST). When adipose-derived stem cells are cultured at about 37°C using a temperature-responsive culture dish, the cells grow well on the hydrophobic culture surface and form a sheet of one layer. Once the sheet is formed, the temperature-responsive culture dish is removed from the incubator and the temperature is lowered to below the lower critical solution temperature (e.g., on ice), and the cell sheet detaches from the culture surface while maintaining the sheet structure. The detached cell sheet can then be collected. The recovery of the sheet can be carried out using a support. The support is not particularly limited, but examples thereof include nonwoven fabric sheets, such as polymer-coated polylactic acid nonwoven fabric sheets. Examples of temperature-responsive polymers include poly(N-alkylacrylamide), poly(N-vinyl alkylamide), polyvinyl alkyl ether, and polyethylene glycol / polypropylene glycol block copolymers. For example, poly(N-isopropylacrylamide) (PNIPAAm) and poly(2-alkyl-2-oxazoline) can be preferably used. In addition, various methods for controlling the LCST (for example, a method of copolymerizing n-butyl methacrylate or a method of copolymerizing N,N-dimethylacrylamide) have been developed, and those skilled in the art can obtain polymers having a desired LCST (for example, 10°C to 35°C). When detaching the cell sheet from the temperature-responsive culture dish, it is not necessary to use a protease such as trypsin, and by not using a protease, the cell sheet can be recovered in a state where the extracellular matrix such as adhesive proteins on the surface is maintained.

[0027] The cell sheet containing the adipose-derived stem cells of the present invention may be provided in a state in which it is placed on a support. The cell sheet containing the adipose-derived stem cells of the present invention may be contained in a transport container. The cell sheet containing the adipose-derived stem cells of the present invention may be contained in a transport container in a state in which it is placed on a support. The transport container can introduce the culture solution in a liquid-tight manner, and preferably can introduce the culture solution in a liquid-tight manner without introducing air bubbles.

[0028] In the present invention, the graft is a transplant material. The graft may be a transplant material that can be used for tendon reconstruction and / or ligament reconstruction. A graft used for such purposes is called a "graft for tendon or ligament reconstruction". The cell sheet containing the adipose-derived stem cells of the present invention can be attached to the surface of the graft and used. In this way, various advantageous effects as described above can be obtained. Various grafts can be used for tendon or ligament reconstruction. For example, tendon or ligament grafts such as hamstring tendon (semitendinosus tendon), patellar tendon, and iliotibial band can be used as grafts for anterior cruciate ligament reconstruction. These grafts can be used in combination with (attached to, for example, attached in sheet form) a cell sheet containing the adipose-derived stem cells of the present invention. The graft can be an autologous graft. This is because an autologous graft is less likely to cause immune-related problems such as immune rejection.

[0029] In the present invention, the graft can be transplanted to the damaged area of ​​the tendon or ligament. The reconstruction of the tendon or ligament can be performed by a physician as appropriate. The cell sheet containing the adipose-derived stem cells of the present invention can be attached to the graft (for example, attached in a sheet form) and then used for the reconstruction of the tendon or ligament (for example, reconstruction of the anterior cruciate ligament). The damaged tendon or ligament can be resected and the graft can be transplanted as a replacement for it to reconstruct the tendon or ligament. A bone hole can be made to fix the graft to the tibia and femur, and the graft to which the cell sheet is attached can be inserted into the inside of the hole. The fixation of the graft to the bone can be performed by suturing the periosteum and the graft or by fixing using an implant. In this case, as in the examples, the cell sheet can be attached to the graft so that the cell sheet enters the bone hole. In addition, if necessary, after these procedures, a cell sheet containing the adipose-derived stem cells of the present invention can be further attached to the graft before closing (attached in a sheet form on the surface or wrapped around it). This is because it is considered that the effect of the cell sheet can be enhanced. Therefore, the cell sheet containing the adipose-derived stem cells of the present invention can be used to attach to a graft fixed to a recipient after transplantation. After transplantation, the recipient can be managed in the same manner as after normal tendon or ligament reconstruction surgery. The recipient is an individual (e.g., a human) to which the graft is transplanted.

[0030] The cell sheet containing the adipose-derived stem cells of the present invention may be provided in a state of being attached to a graft. Alternatively, the cell sheet containing the adipose-derived stem cells of the present invention may be packaged separately from the graft in a single container. When the graft is an autologous transplant, the cell sheet containing the adipose-derived stem cells of the present invention may be provided alone.

[0031] The present invention provides a method for reconstructing a tendon or ligament in a subject in need thereof, comprising fixing a graft to a site in the subject where the tendon or ligament needs to be reconstructed. The present invention provides a method for reconstructing a tendon or ligament in a subject in need thereof, comprising fixing a graft to a site in the subject where the tendon or ligament needs to be reconstructed, wherein the fixed graft has a cell sheet containing adipose-derived stem cells on its surface. The method of the present invention can be performed in the reconstruction of anterior cruciate ligament. The reconstruction of anterior cruciate ligament can include attaching a cell sheet containing adipose-derived stem cells of the present invention to the graft before or after fixation, or before and after fixation. The method of the present invention may further include attaching (e.g., wrapping) the cell sheet to the graft before fixing the graft, or attaching (e.g., wrapping) the cell sheet to the graft after fixing the graft. A transplant material containing a cell sheet containing adipose-derived stem cells can be used in such a method. A transplant material containing a cell sheet containing adipose-derived stem cells can be attached to or wrapped around the graft.

[0032] The present invention provides use of a cell sheet containing adipose-derived stem cells in the manufacture of a transplant material used in the method of the present invention for reconstructing a tendon or ligament. The present invention provides a cell sheet containing adipose-derived stem cells for use in the reconstruction method of the present invention.

[0033] The present invention provides a container containing a cell sheet containing adipose-derived stem cells. The container may be a transport container. In the container, the cell sheet may be held on a support. Alternatively, the cell sheet may be attached to the inner surface of the container while maintaining the sheet shape, and the attachment surface may be coated with a temperature-responsive polymer. The container may be capable of maintaining the cell sheet at 35 to 37°C. The cell sheet may be used for the reconstruction of tendons or ligaments. EXAMPLES

[0034] Study design Eighty-eight 20-week-old female Japanese white rabbits underwent anterior cruciate ligament (ACL) reconstruction in the right knee, and were followed up for 2, 4, 8, 16, and 24 weeks with or without an adipose-derived stem cell (ADSC) sheet. Six cases of complications of patellar dislocation, five cases of poor bone tunnel position, three cases of intraoperative graft creation problems, one case that died during the procedure, and one case of biomechanical experiment problems were excluded, and a total of 72 rabbits were analyzed. Forty-four rabbits were used for biomechanical testing and μCT image analysis (six of which at 24 weeks were also used for electron microscopy analysis), 24 rabbits for histopathological analysis, and four rabbits for immunohistochemical analysis.

[0035] rabbit All rabbits were purchased from Japan Clare (Tokyo, Japan) and kept in a specific pathogen-free environment in an animal facility certified by the Keio University Institutional Animal Care and Use Committee. Rabbits were kept in an environment that complies with the Keio University Animal Experiment Guidelines. All animal experimental protocols were approved by the Keio University Institutional Animal Care and Use Committee and were carried out in accordance with the guidelines approved by the committee.

[0036] Adipose-derived stem cell (ADSC) sheet The cell type used was ADSC (Stem Pro human adipose-derived cell, Lot # 1001002; Invitrogen). At the P4 stage, 4 × 10 5 The ADSCs were seeded on the culture surface and cultured for 4 days until they became overconfluent. The dish was then cooled to room temperature, and the cells were detached from the culture surface as an ADSC sheet. During culture, magnesium ascorbyl phosphate n-hydrate (Fujifilm Wako Pure Chemical Corporation) was added to 0.1 mg / ml medium (MesenPro RS medium; Thermo Fisher).

[0037] Surgical method Ketamine hydrochloride (Daiichi Sankyo Co., Ltd.) 35 mg / kg and xylazine hydrochloride (Bayer) 5 mg / kg were administered subcutaneously in the posterior neck. Buprenorphine 0.2 mg (Otsuka Pharmaceutical Co., Ltd.) was inserted anally, and the antibiotic enrofloxacin (Bayer) 7 mg / kg was administered intramuscularly in the right upper arm. The area was shaved, disinfected, draped, and local anesthesia was administered with lidocaine hydrochloride (Maruishi Pharmaceutical Co., Ltd.) immediately before the procedure. A 5 cm midline incision was made, the medial fascia was incised 1 cm, and the semitendinosus muscle (ST) was identified and a 3-4 cm long graft was harvested. After the muscle was trimmed, baseball sutures were made on both ends of the graft with 4-0 Vicryl Plus (Johnson & Johnson). In the sheet group, a single sheet of approximately 1 cm was then wrapped around the center of the graft. The joint was exposed via the midvastus approach, the patella was laterally dislocated, and the original ACL was resected. A 2.0 mm drill was used to create bone holes in both the tibia and femur in the original foot print of the ACL, and the bones were cleaned. The graft was passed from inside the joint to outside the joint, due to concerns that the sheet would be damaged when the graft passed through the bone hole (Figure 1). The grafts in both the femur and tibia were sutured to the periosteum with 4-0 baseball sutures and sutured under neutral tension. In the sheet group, another sheet was wrapped around the intra-articular graft just before wound closure. After confirming that there was no restriction in the range of motion, the wound was closed, and the skin was covered with dermabond advanced (Johnson and Johnson) to complete the surgery. After surgery, the femur and tibia were connected by the graft and cell sheet, as shown in Figure 1, and the cell sheet was brought into contact with the bone hole. The weight change of the individual after surgery and the length and diameter of the graft after transplantation were as shown in Figure 2. As shown in Figure 2, no significant difference was observed between the graft transplantation group with a sheet (sheet group) and the graft transplantation group without a sheet (control group).

[0038] Histological evaluation After sacrifice, specimens for pathological analysis were fixed in 10% neutral formalin for three days and decalcified with EDTA for five weeks. Then, tissue sections were prepared by cutting the femoral tunnel along the axis of the tunnel and stained with Masson's trichrome. Histological evaluation of the healing process of the tendon-bone junction and graft parenchyma was performed using a bright-field microscope. The scoring system used was the same as that of Liu, which has been previously reported. 25,43 (See Table 1.) A notable feature of Liu's scoring system is that it allows for the evaluation of the degree of maturity of the tendon-bone interface.

[0039] [Table 1]

[0040] Micro-computed tomography Imaging of the femur was performed immediately before the biomechanical experiment. μCT was performed using an R_mCT2 (Rigaku Corporation) under the following conditions: fields of view: 30 mm, voltage: 90 kV, current: 160 mA, scan technique: fine for 3 min. The bone volume / tissue volume (%, BV / TV) and bone tunnel cross-sectional area were determined using a 3D BON system (Ratoc) and ImageJ software (ImageJ version 1.52; National Institutes of Health). The bone tunnel cross-sectional area was determined by dividing the femoral tunnel into four parts, defining the part closest to the articular surface as the articular opening, and measuring the cross-sectional area at this site. BV / TV was measured as the bone mineral content in a 3.5 mm cube at the center of the femoral tunnel (Figure 2).

[0041] Biomechanical Experiments Immediately after sacrificing the individual, the soft tissues around the knee were removed, leaving only the bone and graft, which were then frozen and stored at -80°C. Before testing, the specimens were thawed at room temperature, and a maximum failure load test was performed at a speed of 10 mm / min using an Autograph AG-IS tensile tester (Shimadzu Corporation), with the k-wires passed through the tibia and femur and set on the machine stand (see Figure 10). Based on the curve results obtained from the experiment, the ultimate failure load (UFL) and stiffness were measured.

[0042] Transmission electron microscope Scanning type After biomechanical analysis, the intact grafts were fixed with 2% paraformaldehyde (PFA) and 2% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB) pH 7.4, overnight at 4°C. After this fixation, the samples were washed three times for 30 min each in 0.1 M PB and post-fixed overnight at 4°C in 2% osmium tetroxide (OsO4) in 0.1 M PB. The obtained samples were dehydrated in graded ethanol solutions. After these dehydration steps, the samples were serially dehydrated in 100% ethanol overnight at room temperature. The obtained samples were infiltrated twice with propylene oxide (PO) for 1 h each and placed in a 70:30 mixture of PO and resin (Quetol-812; Nissin EM Co., Ltd.) for 2 h, after which the tube caps were left open to allow PO to volatilize overnight. After transferring to fresh 100% resin and polymerizing at 60 °C for 48 h, the blocks were ultrathin sectioned at 70 nm with a diamond knife using an ultramicrotome (ULTRACUT UCT; Leica) and the sections were placed on copper grids. The grids were observed in a transmission electron microscope (JEM-1400Plus; JEOL Ltd.) at an accelerating voltage of 100 kV. Digital images (3296 × 2472 pixels) were captured with a CCD camera (EM-14830RUBY2; JEOL Ltd.). Ten TEM images were taken randomly in a blinded manner for each sample and analyzed using an image analysis system (ImageJ version 1.52; National Institutes of Health). For the evaluation of fibril diameter, the smallest fibril diameter was always measured to prevent errors due to oblique sectioning. Fibril structures with diameters <10 nm were excluded to avoid measuring microfibrils or non-collagen fibril components. The number of collagen fibers, fibril diameter, and collagen occupancy / field were calculated.

[0043] RNA expression analysis One knee joint was taken from each of the control and sheet groups at 4 weeks after surgery, and only the intra-articular graft portion was collected. The collected graft was immediately soaked in an appropriate amount of RNA later, and then minced with 1 mL of Trisol for 3 minutes. After mixing with 200 μL of chloroform, the mixture was vortexed and centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant was transferred to a new microtube, and 500 μL of 2-propanol was added. 700 μL was transferred to an RNA Mini kit (Qiagen) column and centrifuged at 12,000 rpm at room temperature (RT) for 15 seconds. 350 μL of RW1 was added, and the mixture was centrifuged at 12,000 rpm at RT for 15 seconds. Next, 10 μL of DNase (RNase-free DNase Set; Qiagen) + 70 μL of RDD was dropped onto the column membrane and left at room temperature for 15 minutes. The samples were then washed three times by centrifugation using 500 μL of RPE. They were then centrifuged for 2 minutes using RPE. The samples were stored at -80°C. Genewiz was commissioned to carry out expression analysis of the samples based on the number of reads from sequencing. The gene expression level in the case of the sheet (Sheet(+)) was calculated with the control (Sheet(-)) set to 1.

[0044] statistical analysis Bar and line graph data were expressed as mean ± SD. Boxplot values ​​are shown as minimum, maximum, sample median, and first and third quartiles. Statistical analysis was performed using Welch's t-test between the control and sheet groups at each time point. Pearson's correlation analysis was performed for correlations between biomechanical tests and BV / TV, and ANOVA test was used for electron microscopy analysis. A P value <0.05 was considered statistically significant.

[0045] [result] In the histopathological evaluation, as shown in Figures 3 to 6, the grafts went through the commonly known necrosis (inflammation) stage, followed by the regrowth stage, and then the maturation (remodeling) stage. Specifically, at 4 weeks (4w) after graft implantation, the number of cells in the graft parenchyma decreased in both the sheet group and the control group, and the arrangement of collagen fibers became irregular. On the other hand, compared to the control group, the sheet group had a relatively higher number of fibroblasts, and the number of fibroblasts peaked at that point. In other words, the control group showed a tissue image of the necrosis stage, but the sheet group showed a tissue image of the necrosis stage as well as a partial regrowth stage. At the tendon-bone junction, the control group also showed the formation of Sharpey's-like fibers and the appearance of hypertrophied chondrocytes, but these were more common in the sheet group. The sheet group also showed less bone destruction. At 8 weeks (8w), the arrangement of collagen fibers in the graft stroma was still irregular in both the sheet and control groups. The graft stroma in the control group had entered the repopulation phase, with the number of fibroblasts increasing and peaking compared to 4 weeks, whereas in the sheet group, the number of cells tended to decrease compared to 4 weeks, and the repopulation phase was nearing its end. Sharpey's-like fibers and hypertrophied chondrocytes were clearly observed at the tendon-bone junction in the control group, whereas osteoid formation had begun to be observed in the sheet group, and there was less destruction at the junction than in the control group. At 16 weeks (16w), the graft areas in both the sheet group and the control group had entered a remodeling phase, and although the arrangement of cells within the graft was still irregular, it gradually began to assume a spindle shape, the number of cells within the graft tended to decrease compared to 8 weeks, and the arrangement of collagen fibers had become more regular compared to up to 8 weeks. Osteoid formation was evident at the tendon-bone junction, even in the control group, and in both groups the tendon-bone junction had formed a smooth junction with few irregularities and an unclear boundary. At 24 weeks (24w), the number of cells in the graft parenchyma in both the sheet group and the control group was comparable to that of the original ST, and compared to before 16 weeks, the cells were more regularly arranged, and the collagen fibers were oriented in a similar manner to the original graft. Furthermore, the tendon-bone junction maintained an unclear boundary, just as it had at 16 weeks, and lamellar bone formation was observed.

[0046] When the above was evaluated using the scoring system described above, as shown in Figure 4, the control group vs. sheet group had 3.0±0.9 vs. 4.2±0.8 (P=0.04) after 4 weeks, 5.7±0.5 vs. 6.5±0.5 (P=0.02) after 8 weeks, 8.2±0.4 vs. 8.5±0.5 (P=0.26) after 16 weeks, and 8.7±0.5 vs. 9.0±1.1 (P=0.52) after 24 weeks.

[0047] Micro-computed tomography evaluation The femoral tunnel was divided into four sections, and the section closest to the joint was defined as the joint opening. The cross-sectional area (mm 2 ) were measured. The results are shown in Figure 7. As shown in Figure 7, in the control group (-) vs. the sheet group (+), the results were 3.9±1.1 vs. 3.5±1.1 (P=0.61) after 2 weeks, 7.3±1.0 vs. 5.1±0.9 (P=0.01) after 4 weeks, 6.6±2.1 vs. 4.7±0.8 (P=0.25) after 8 weeks, 4.3±0.7 vs. 4.6±1.0 (P=0.64) after 16 weeks, and 4.3±1.1 vs. 4.1±1.5 (P=0.53) after 24 weeks. The cross-sectional area of ​​the intra-articular opening was significantly smaller in the sheet group at 4 weeks after surgery, but no significant differences were observed at other time points. In addition, to evaluate the calcification of the tendon-bone junction, the area with a calcification degree of 400 mg / ml or more was calculated as bone, and BV / TV (%) was calculated. The results are shown in Figure 8. As shown in the left panel of Figure 8, in the control group (-) vs. the sheet group (+), the calcification degree of the bone at the tendon-bone junction increased over time, as shown in the following chart. After 2 weeks, the calcification degree was 21.9 ± 6.0 vs. 20.1 ± 5.9 (P = 0.65), after 4 weeks, the calcification degree was 21.5 ± 3.7 vs. 22.8 ± 2.0 (P = 0.53), after 8 weeks, the calcification degree was 29.5 ± 1.3 vs. 24.4 ± 3.5 (P = 0.06), after 16 weeks, the calcification degree was 33.5 ± 2.6 vs. 38.4 ± 4.6 (P = 0.11), and after 24 weeks, the calcification degree was 32.4 ± 4.4 vs. 43.4 ± 8.2 (P = 0.05). When the area with a mineralization degree of 800 mg / ml or more was analyzed as bone, the results were 0.41 ± 0.2 vs. 0.33 ± 0.2 (P = 0.56) after 2 weeks, 0.7 ± 0.7 vs. 1.3 ± 0.8 (P = 0.29) after 4 weeks, 1.2 ± 0.8 vs. 2.6 ± 2.2 (P = 0.35) after 8 weeks, 3.3 ± 1.8 vs. 6.8 ± 4.6 (P = 0.21) after 16 weeks, and 4.0 ± 3.4 vs. 10.0 ± 1.8 (P = 0.02) after 24 weeks, as shown in the right panel of Figure 8. For all mineralization degrees, BV / TV was significantly higher in the sheet group at 24 weeks.

[0048] Biomechanical Experiments The results of the maximum breaking load test for the control group (sheet-) and the sheet group (sheet+) are shown in Figure 10A. As shown in Figure 10A, the maximum breaking load (N) was 17.2 ± 5.5 vs. 37.3 ± 10.3 (P = 0.01) after 2 weeks, 28.6 ± 1.9 vs. 47.4 ± 10.4 (P = 0.003) after 4 weeks, 53.0 ± 14.3 vs. 48.1 ± 9.3 (P = 0.59) after 8 weeks, 66.2 ± 9.3 vs. 95.2 ± 43.1 (P = 0.24) after 16 weeks, and 66.7 ± 27.3 vs. 85.3 ± 29.5 (P = 0.39) after 24 weeks. In the biomechanical experiment, the maximum breaking load was higher in the sheet group except for the 8th week, and a significant difference was observed especially in the early period up to the 4th week. The stiffness values ​​(N / mm) were as shown in Figure 10B. The stiffness values ​​were as follows: 7.7 ± 0.7 vs 13.1 ± 2.5 (P = 0.01) after 2 weeks, 14.6 ± 4.5 vs 18.0 ± 5.3 (P = 0.31) after 4 weeks, 17.5 ± 5.8 vs 16.2 ± 4.9 (P = 0.74) after 8 weeks, 24.8 ± 7.0 vs 28.7 ± 6.0 (P = 0.43) after 16 weeks, and 24.0 ± 4.2 vs 26.5 ± 6.0 (P = 0.52) after 24 weeks. No significant differences were observed in stiffness values ​​except for 2 weeks after surgery.

[0049] The fracture patterns in the biomechanical experiment were as shown in Figure 11: the graft pulled out from the tibia (left panel of Figure 11), the graft pulled out in the middle (center of Figure 11), and the graft pulled out from the femur (right panel of Figure 11). The number of fractures in the middle and the graft pulled out from the bone are shown in Table 2. The results showed that the rate of parenchymal fractures was higher in the sheet group at each week (Table 2).

[0050] [Table 2]

[0051] When we examined the correlation between the degree of calcification at the tendon-bone junction (400 mg / ml) and the breaking load of specimens that had experienced tendon detachment from the bone, excluding cases where parenchymal rupture had occurred, we found a correlation of R = 0.72 (P = 0.0002) (Figure 12).

[0052] Evaluation by transmission electron microscope The total number of fibers per field of view was 333±125 in the original ST, whereas in the electron microscopy analysis 24 weeks after surgery, the number was 527±235 vs. 473±175 in the control group (-) vs. the sheet group (+), with no significant difference (Fig. 9). However, while the fiber diameter (minimum ferret diameter; nm) was 90±16 in the original ST, in the electron microscopy analysis 24 weeks after surgery, the fiber diameter was 50.5±51.5 vs. 58.7±56.8 (P< 0.001) in the control group (-) vs. the sheet group (+), with the sheet group being significantly thicker (Fig. 9, lower left panel). Looking at the histogram of fiber diameter, the sheet group had a composition closer to the ST (Fig. 9, lower center panel). Furthermore, while the percentage of collagen fibers per field of view was 64.0±1.3 in the original ST, electron microscopy analysis 24 weeks after surgery showed that it was 47.4±14 in the control group (-) vs. 54.5±11 in the sheet group (+) (P=0.03), meaning that it was significantly higher in the sheet group (Figure 9, lower right panel).

[0053] The results of gene expression analysis are shown in Figures 13 and 14. As shown in Figure 13, the group in which the graft was transplanted together with the cell sheet showed higher expression than the control group for IL-1β, PDGF-A, and VEGF-A.

[0054] According to this example, the number of ADSC cells in the ADSC sheet wrapped around the graft was advantageously smaller than the number of ADSC cells in the suspension. The reason for this is thought to be that when the cell suspension was injected, the transplanted cells were dispersed throughout the joint, resulting in a decrease in cell concentration at the required site. In this experiment, ADSCs were wrapped around the graft in the form of a sheet to clarify the localization of the cells, but the ADSC sheet has an extracellular matrix formed on its surface, which is different from ADSCs in a suspension.

[0055] According to this example, bone tunnel enlargement was significantly prevented in the sheet group 4 weeks after surgery compared to the control group. It has been reported that bone tunnel enlargement occurs after surgery in ACL reconstruction using the hamstrings, causing the graft to loosen, requiring reoperation. 9,47 In addition, in ACL reconstruction surgery, the bone tunnel temporarily expands at a relatively early stage. 48 It has also been reported that the condition may shrink over time. 25,29 However, there are also reports that transplantation of MSCs can prevent bone tunnel enlargement. 15 In the present experiment, the action of ADSCs suppressed the postoperative transient bone destruction and bone resorption at the tendon-bone junction at 4 weeks, preventing the enlargement of the bone tunnel and increasing the contact area between the bone and the graft, which may have resulted in a significant improvement in the maximum breaking load at an early stage in the biomechanical experiment, except for the 8th week.

[0056] Pathologically, the transplanted grafts underwent a similar process to the tissue repair reported by Kosaka. 21 In the sheet group, fibroblast migration into the graft stroma occurred at a relatively early stage, angiogenesis was greater than in the control group, and Sharpey's-like fiber formation and hypertrophic chondrocytes were more prevalent. At 4 weeks, the sheet group partially developed a tissue image similar to that of the control group at 8 weeks, and the transplantation of ADSCs promoted the maturation of the graft stroma and tendon-bone junction, which is thought to be one of the factors that led to the improvement in biomechanical strength in the sheet group.

[0057] It is possible that the action of trophic factors from ADSCs promoted the recovery of the tendon-bone junction, or that ADSCs themselves functioned as living cells in the graft parenchyma. In this case, the presence of an extracellular matrix (adhesive protein) in the ADSC sheet may have had a positive effect on the results.

[0058] Furthermore, biomechanical experiments showed that the rate of parenchymal ruptures increased over time in both groups, but in the sheet group, ADSCs promoted the formation of bone tissue with a particularly high degree of mineralization, and it is believed that the increase in the degree of mineralization at the tendon-bone junction over time reduced the rate of tendon detachment from the bone. In a report in which ADSC gel transplantation was used in conjunction with surgery for chronic rotator cuff tears, it was reported that BMD (bone mineral density) at the tendon-bone junction improved. 17 This experiment also suggested that ADSCs have bone formation activity.

[0059] After 16 weeks, pathologically, the boundary at the tendon-bone junction became unclear in both groups, and lamellar bone structure gradually became visible, but there was no significant difference in the scores. Furthermore, no significant difference was observed in bone hole enlargement between the two groups after 16 weeks. However, in the biomechanical experiment, the average maximum breaking load was higher in the sheet group compared to the control group, so a transmission electron microscope analysis was performed to evaluate the parenchyma. According to a report by Takeuchi, the collagen fibers in transplanted grafts shrink over time, and the percentage of collagen fibers occupying the area also decreases. 46 It has also been reported that many of the newly synthesized fibers after surgery are thin and made of type 3 collagen. 33 The results of this electron microscope study suggest that ADSCs promoted the synthesis of type 1 collagen and inhibited the synthesis of type 3 collagen, which resulted in thicker collagen fiber diameters in the sheet group than in the control group and a higher collagen fiber occupancy rate per cross-sectional area, which may have contributed to an improvement in the maximum breaking load in the event of a parenchymal rupture. Previous reports have shown that re-ruptures after ACL reconstruction surgery occur more frequently in the parenchyma than in the tendon-bone junction. 28,38By using ADSCs in combination, it is expected that the re-rupture rate can be reduced by strengthening the parenchymal part.

[0060] TIFF0007654205000003.tif209144TIFF0007654205000004.tif208144TIFF0007654205000005.tif208146TIFF0007654205000006.tif203145

Claims

1. A transplant material for reconstruction of the anterior cruciate ligament, comprising a cell sheet containing adipose-derived stem cells, the cell sheet maintaining adhesive proteins on the surface of the cell sheet, and the cell sheet containing 90% or more cells positive for CD29, CD44, CD73, CD90, CD105, and CD166, and 5% or less cells positive for CD14, CD31, CD45, and Lin1.

2. The transplant material according to claim 1 , comprising a stratified cell sheet.

3. 3. The transplant material according to claim 1, which is used by being attached to the surface of the junction between the ligament and the bone of a graft for reconstruction of the anterior cruciate ligament.

4. 3. The transplant material according to claim 1, which is used by being attached to a surface other than the junction between the ligament and the bone of a graft for reconstruction of the anterior cruciate ligament.

5. A product for reconstruction of the anterior cruciate ligament comprising a container containing the implant material according to any one of claims 1 to 4.

6. A transplantation material comprising the cell sheet defined in any one of claims 1 to 4, for use in transplanting in an attached state onto a transplant graft, thereby promoting the production of type 1 collagen in the graft.

7. A transplant material comprising the cell sheet defined in any one of claims 1 to 4, for use in transplanting in a state of being attached to a transplant graft, thereby suppressing the production of type 3 collagen in the graft.

8. The implant is attached to the graft, which allows the collagen fibers in the graft to be attached. A transplant material comprising a cell sheet as defined in any one of claims 1 to 4 for use in increasing the diameter of a tissue.

9. A transplant material comprising the cell sheet defined in any one of claims 1 to 4, for use in transplanting in a state of being attached to a transplant graft, thereby improving the occupancy rate of collagen fibers in the cross section of collagen fibers in the graft.

10. A transplant material comprising the cell sheet defined in any one of claims 1 to 4, which is transplanted in a state of being attached to a transplant graft, thereby for use in suppressing rupture of the graft after transplantation.

11. A transplant material comprising the cell sheet defined in any one of claims 1 to 4, which is transplanted in a state of being attached to a transplant graft, thereby increasing the collagen fiber diameter in the graft, improving the occupancy rate of collagen fibers in the collagen fiber cross section in the graft, promoting the production of type 1 collagen in the graft, and suppressing the production of type 3 collagen in the graft.

12. The implant material of any one of claims 1 to 4, further comprising a transplant graft.

Images