Complex for bone regeneration or augmentation, and method for producing same

Abstract

The present invention provides a complex for bone regeneration or augmentation, the complex comprising a bone substitute material covered with a layered cell sheet containing osteogenic cells.

Description

Bone regeneration or augmentation composite and method for producing the same

[0001] The present invention relates to a composite for bone regeneration or augmentation and a method for producing the same, and more specifically to a composite for bone regeneration or augmentation obtained by covering a bone filler with a stratified cell sheet containing osteogenic cells, and a method for producing the same.

[0002] In the fields of orthopedics and dentistry, there have been intensive studies into the development of methods for regenerating or augmenting bones lost due to accidents, diseases, or congenital abnormalities (Patent Documents 1 to 3).

[0003] Autologous bone grafting has been the gold standard in conventional bone regeneration therapy. However, because autologous bone grafting requires advanced techniques and complex equipment to harvest, it is extremely difficult to perform safely and easily. In addition, it is highly invasive and places a significant burden on patients, making it a treatment that requires improvement. Therefore, there is a great need for alternative methods to autologous bone grafting.

[0004] As alternatives to autologous bone transplantation, methods that are being considered include using biocompatible materials such as hydroxyapatite as bone fillers, using osteogenic cells (mesenchymal stem cells, osteoblasts, etc.) that are involved in bone formation in the body, and using growth factors such as basic fibroblast growth factor (bFGF) and bone morphogenetic protein (BMP).

[0005] However, these alternative approaches also have their own challenges. For example, methods using biocompatible materials as bone fillers have the problem that bone regeneration takes a relatively long time because the bone filler implanted into the body does not actively act on biological tissues. Furthermore, such bone fillers are typically very small granular materials (approximately 0.15 to 2 mm), making them difficult to handle. Furthermore, methods using osteogenic cells require the proliferation of autologous cells, which requires significant time and cost to prepare the required number of cells. Furthermore, methods using growth factors also have problems such as the inability to precisely control bone regeneration, high manufacturing costs, and variable effects depending on the patient's health condition.

[0006] In addition, a common problem with these techniques is the difficulty of horizontal / vertical bone augmentation.

[0007] Specifically, in bone regeneration and augmentation, it is relatively easy to regenerate or augment bone defects resulting from bone marrow cavities caused by tooth extraction sockets, fractures, etc. However, it is extremely difficult to increase bone width (i.e., horizontal bone augmentation) or bone height (i.e., vertical bone augmentation) in areas of extensive bone defects caused by accidents, diseases, congenital abnormalities, etc. using conventional bone augmentation methods.

[0008] Therefore, there is a strong demand for the establishment of a new method for bone regeneration / augmentation that can solve the problems of the conventional techniques.

[0009] WO2014 / 115562WO2014 / 017147WO2020 / 226043

[0010] An object of the present invention is to provide a novel bone regeneration / augmentation tool that enables horizontal / vertical bone regeneration or augmentation and has good operability, and also to provide a method for easily and efficiently manufacturing the tool.

[0011] As a result of extensive research into the above-mentioned problems, the inventors have found that a composite obtained by covering a bone filler with a stratified cell sheet containing mesenchymal stem cells (1) enables horizontal and vertical bone augmentation by transplanting a composite of an appropriate size for the size of the bone defect, (2) when transplanted into a living body, new bone is formed not only on the host bone side but also in the center of the composite, (3) when transplanted into a living body, in addition to bone formation, bone marrow-like tissue is also formed, thereby reproducing a structure similar to bone tissue present in the living body, and (4) when a composite is produced using only two elements, a bone filler and a stratified cell sheet, it can be produced using a very simple process, and as a result, composites of uniform quality can be produced efficiently and easily. Furthermore, the inventors have found that, particularly when using a granular bone filler, (5) operability is significantly improved by covering the fine particulate bone filler with a stratified cell sheet to form a roughly spherical composite, (6) a bone regeneration / augmentation composite of a desired size can be prepared by adjusting the amount of bone filler and the area of ​​the stratified cell sheet, and (7) because the composite is roughly spherical, the orientation of the composite is not limited during transplantation. Based on these findings, the inventors have conducted further research and have completed the present invention. Specifically, the present invention is as follows.

[0012] [1] A composite for bone regeneration or augmentation, comprising a bone filler coated with a stratified cell sheet containing osteogenic cells. [2] The composite of [1], wherein the bone filler is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite. [3] The composite of [1] or [2], wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells. [4] A method for producing a composite for bone regeneration or augmentation, comprising the following steps: coating a bone filler with a stratified cell sheet containing osteogenic cells. [5] A method for producing a composite for bone regeneration or augmentation according to [4], comprising the step of preparing a stratified cell sheet containing osteogenic cells prior to the step of coating a bone filler with a stratified cell sheet containing osteogenic cells. [6] The manufacturing method according to [4] or [5], wherein the bone filler is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite. [7] The manufacturing method according to any one of [4] to [6], wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells. [8] A kit for manufacturing a composite for bone regeneration or augmentation, comprising a bone filler and a stratified cell sheet containing osteogenic cells. [9] The kit according to [8], wherein the bone filler is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

[10] The kit according to [8] or [9], wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

[11] A stratified cell sheet containing osteogenic cells for use in combining with a bone filler to manufacture a composite for bone regeneration or augmentation.

[12] The stratified cell sheet containing osteogenic cells according to

[11] , wherein the bone filler is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

[13] The stratified cell sheet according to

[11] or

[12] , wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

[14] A bone filler for use in producing a composite for bone regeneration or augmentation by combining with a stratified cell sheet containing osteogenic cells.

[15] The bone filler according to

[14] , wherein the bone filler is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

[16] The bone filler according to

[14] or

[15] , wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

[0013] According to the present invention, it is possible to treat a wide range of bone defects requiring horizontal / vertical bone regeneration and / or bone augmentation. Furthermore, according to the present invention, it is possible to efficiently prepare a novel composite for bone regeneration or augmentation that enables such treatment. In particular, although it has been reported that layered transplanted cells are unable to remain in the local area and migrate to other sites or die, this example demonstrated that the transplanted cells not only survived but also differentiated into osteocytes even 12 weeks after transplantation. This is thought to be due to the paracrine / autocrine effect of cytokines secreted by the surviving cells, which are maintained in an environment conducive to cell survival by the layered cells and extracellular matrix.

[0014] FIG. 1 is a diagram showing one embodiment of the composite of the present invention. FIG. 2 is a diagram showing the results of transplanting a composite of the present invention prepared using rat femur-derived bone marrow mesenchymal stem cells into the parietal region of a rat. (a) shows the implantation site of the composite of the present invention. (b) shows the results of HE staining of the implantation site 12 weeks after implantation. FIG. 3 is a diagram showing the results of transplanting a composite of the present invention prepared using human iliac bone marrow mesenchymal stem cells into an immunodeficient mouse. (a) shows the results of HE staining and immunostaining for human-specific Vimentin of the implantation site 12 weeks after implantation. (b) shows the percentage of new bone formation in the control and the composite of the present invention (human iliac MSCs). FIG. 4 is a diagram showing the appearance of three composites of the present invention of different sizes prepared in 35 mm dishes, 60 mm dishes, and 90 mm dishes. FIG. 5 is a diagram showing the results of HE staining of the implantation site 12 weeks after implantation when composites of different sizes of the present invention were implanted into the parietal region of a rat. FIG. 6 shows the results when complexes of the present invention of different sizes were transplanted into rats under the condition that the number of cells present on the surface of the complex was uniform.

[0015] The present invention will be described in detail below.

[0016] 1. Composite for bone regeneration or augmentation The present invention provides a composite for bone regeneration or augmentation (hereinafter sometimes referred to as the "composite of the present invention"), which comprises a bone filler coated with a stratified cell sheet containing osteogenic cells.

[0017] As used herein, the term "bone prosthetic material" refers to a biocompatible and osteoconductive material used for bone regeneration and augmentation in the fields of orthopedics and dentistry. "Bone prosthetic material" includes autologous bone or processed materials derived therefrom, allogeneic bone or processed materials derived therefrom, xenogenic bone or processed materials derived therefrom, and artificial bone. In the present invention, these bone prosthetic materials may be used alone or in combination of two or more. In a preferred embodiment of the present invention, the bone prosthetic material may be artificial bone. Examples of artificial bones that can be used in the present invention include, but are not limited to, hydroxyapatite (HA), β-tricalcium phosphate (β-TCP), and carbonate apatite (CAP). Bone prosthetic materials that can be used in the composites of the present invention can be prepared by known methods, or commercially available products may be used. Commercially available bone prosthetic materials include, for example, HA, such as "APACERAM" (HOYA Technosurgical Corporation), "NEOBONE" (KENTEC Co., Ltd.), and "CALCITITE" (HAKUHO Co., Ltd.). Examples of β-TCP include "OSFERION (registered trademark)" (Olympus Corporation), "CERASOLV (registered trademark)" (GINVI Japan LLC), and "SUPERPORE" (HOYA Technosurgical Corporation). Examples of CAP include "CYTRANS (registered trademark) GRANULES (GC Corporation)."

[0018] The shape of the bone filler used in the composite of the present invention is not particularly limited. The shape of the bone filler may be selected appropriately depending on the type of bone regeneration required. For example, when the objective is to regenerate bone with a relatively complex form, one or more composites of the present invention in which a granular bone filler is coated with a stratified cell sheet can be used, as needed. When the objective is to regenerate bone with a relatively simple form, the composite of the present invention can be used in which a block of bone filler made to fit the intended regeneration site using a CAD / CAM (computer-aided design / computer-aided manufacturing) system or the like is coated with a stratified cell sheet.

[0019] In one embodiment, the bone prosthetic material used in the composite of the present invention is characterized by its granular form. Generally, the granule diameter of granular bone prosthetic materials is approximately 0.15 mm to 2 mm, making it difficult to handle individual granules. However, in the present invention, the granular form allows for adjustment of the size of the composite to be produced, while the layered cell sheet coating ensures ease of handling. The granule diameter of the bone prosthetic material used to produce the composite of the present invention can be, but is not limited to, typically 0.01 mm or greater, preferably 0.1 mm or greater, 0.2 mm or greater, 0.3 mm or greater, and more preferably 0.5 mm or greater. Furthermore, the granule diameter of the bone prosthetic material can be, but is not limited to, typically 10 mm or less, preferably 6 mm or less, 5 mm or less, 4 mm or less, and more preferably 3 mm or less. In one embodiment, the granule diameter of the bone prosthetic material can be, but is not limited to, typically 0.01 to 10 mm, preferably 0.1 to 6 mm, 0.2 to 5 mm, 0.3 to 4 mm, and more preferably 0.5 to 3 mm.

[0020] The composite of the present invention is characterized in that a bone substitute material is coated with a stratified cell sheet containing osteogenic cells. As used herein, "osteogenic cells" refers to cells directly involved in bone formation or their precursor cells. Examples of osteogenic cells suitable for use in preparing the composite of the present invention include, but are not limited to, mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells. The tissue from which mesenchymal stem cells are derived may be any tissue, such as bone marrow, adipose tissue, umbilical cord, placenta, dental pulp, or synovium. Furthermore, the osteogenic cells may be derived from iPS cells.

[0021] As used herein, the term "stratified cell sheet" refers to a structure composed of a large number of cells layered together and an extracellular matrix derived from the cells. A stratified cell sheet differs from a monolayer cell sheet in that at least some of the cells are biologically bonded in three dimensions. A stratified cell sheet with such biological bonds is characterized by the fact that the bonds between cells are not substantially destroyed, and as a result, it can be handled as a sheet. Such biological bonds include bonds between cells via an extracellular matrix. Such a stratified cell sheet does not simply mean a single-layer cell sheet that is simply layered into multiple layers, but refers to a sheet formed so that the cells have a multi-layer structure of two or more layers at the time of cell sheet formation. The number of cells contained in the stratified cell sheet of the present invention is 9.5 cm ２ 3 x 10 per ６ ~10 x 10 ６ cells, 4 x 10 ６ ~8 x 10 ６ cells, 5 x 10 ６ ~6 x 10 ６ Cell, 21cm ２ 8 x 10 per ６ ~14 x 10 ６ cells, 10x10 ６ ~12 x 10 ６ cells, 11 x 10 ６ Cell, 55cm ２ 22 x 10 per ６ ~42 x 10 ６ cells, 27 x 10 ６ ~37 x 10 ６ cells, 32 x 10 ６ Alternatively, the unit area (cm) of the culture vessel can be ２ The number of cells per ５ ~2 x 10 ６ cells, 3 x 10 ５ ~1 x 10 ６ cells, 3 x 10 ５ ~7 x 10 ５ cells, 4 x 10 ５ ~8 x 10 ５ cells, 5 x 10 ５ ~7 x 10 ５ cells, 5 x 10５ ~6 x 10 ５ cells, 5 x 10 ５ cells, or 6 x 10 ５ It may be a cell.

[0022] A known method may be used to prepare a layered cell sheet containing osteogenic cells (e.g., Ando, ​​W. et al., Tissue Eng Part A. 2008;14(12):2041-9.). Briefly, osteogenic cells are seeded in a cell culture vessel at about 20 to about 160 times the usual seeding amount, and cultured in a medium suitable for the cells under an environment of temperature and humidity, for example, 5% CO ２ A layered cell sheet can be prepared by carrying out adhesion culture in a humidified incubator at 37°C until a multilayer structure is formed. ２ ) per 1.0 x 10 ６ ~8 x 10 ６ Cells, preferably 1.8 x 10 ６ ~7.2 x 10 ６ Alternatively, the cells are seeded at 1.0 × 10 cells per unit area of ​​the culture vessel. 5 ~8 x 10 5 cells / cm ２ , preferably 2 × 10 ５ ~8 x 10 ５ cells / cm ２ The medium used is α-MEM containing 10% FBS, 1% antibiotic-antimycotic, and 0.2 mM ascorbic acid, and the cells are cultured for 10 days or more, for example, 10 to 20 days, or 10 to 14 days, to prepare a layered cell sheet.

[0023] In preparing the composite of the present invention, the method for covering the bone filler with a stratified cell sheet containing osteogenic cells is not particularly limited as long as it allows the bone filler to be covered by the stratified cell sheet. Typically, the bone filler is placed in the center of the stratified cell sheet. The edge of the stratified cell sheet is then grasped using tweezers or the like, and the stratified cell sheet is moved so that the bone filler is enveloped by the stratified cell sheet, thereby covering the bone filler with the stratified cell sheet. If necessary, the method may also include a step of first culturing osteogenic cells in a culture dish to prepare a stratified cell sheet. The bone filler does not need to be covered 100% by the stratified cell sheet, as long as the object of the present invention can be achieved. For example, the coverage may be sufficient to maintain the coverage when the composite is grasped with tweezers or a similar grasping tool.

[0024] The shape of the composite of the present invention is not particularly limited, as long as it is a shape that fits the bone defect site. The shape of the composite of the present invention may be, for example, approximately spherical, approximately cylindrical, or approximately rectangular parallelepiped. In a preferred embodiment, the shape of the composite of the present invention is approximately spherical. The approximately spherical shape of the composite of the present invention is expected to provide the same effect regardless of the direction from which it is transplanted into a living body. Furthermore, since the direction is not limited during transplantation, any part of the composite can be grasped with tweezers, which is advantageous in that it is easy to handle. The method for forming the composite into an approximately spherical shape is not particularly limited. The composite may be formed into an approximately spherical shape when the bone prosthetic material is coated with a stratified cell sheet using tweezers or the like, or the bone prosthetic material may be coated with a stratified cell sheet to prepare the composite, and then the shape of the composite may be formed into an approximately spherical shape using tweezers or the like. In this specification, the term "approximately spherical" refers to a shape that can be recognized as a sphere. Specifically, "approximately spherical" includes both perfect spheres and shapes that are not perfect spheres but are recognizable as spheres.

[0025] The composite of the present invention can be prepared in a variety of sizes by adjusting the amount of bone filler and the area of ​​the layered cell sheet. When the composite of the present invention is approximately spherical, its size can typically be, but is not limited to, a major axis of 1 to 30 mm, a minor axis of 1 to 20 mm, a major axis of 2 to 25 mm, a minor axis of 1 to 15 mm, a major axis of 3 to 20 mm, a minor axis of 1 to 10 mm, a major axis of 4 to 15 mm, a minor axis of 2 to 8 mm, a major axis of 6 to 10 mm, a minor axis of 4 to 6 mm, a major axis of 1 to 10 mm, and a minor axis of 1 to 10 mm, preferably a major axis of 1 to 9 mm, a minor axis of 1 to 9 mm, a major axis of 1 to 8 mm, a minor axis of 1 to 8 mm, a major axis of 1 to 7 mm, and a minor axis of 1 to 7 mm, and more preferably a major axis of 1 to 6 mm and a minor axis of 1 to 6 mm. In this specification, the terms "major axis" and "minor axis" refer to the longest and shortest diameters of a roughly spherical body. The composite of the present invention can be prepared by covering one or more block-shaped bone filler materials with a stratified cell sheet, and its size can typically be, but is not limited to, 10 to 100 mm in length, 10 to 40 mm in width, and 5 to 20 mm in height.

[0026] In a preferred embodiment of the present invention, the composite of the present invention is a granular bone substitute coated with a stratified cell sheet, is approximately spherical, has a major axis of 3 to 10 mm or 3 to 5 mm, and a minor axis of 3 to 6 mm or 3 to 5 mm, and the number of osteogenic cells present on the surface of the composite is 1.0 × 10 ６ ~6.0 x 10 ６ The composite having such a size is preferable for carrying out the present invention because it is easy to handle and can be easily grasped with tweezers, can be easily transplanted into a relatively narrow area such as the oral cavity, and allows for horizontal / vertical bone regeneration and / or augmentation. When preparing a composite of such a size, a layered cell sheet having a diameter of about 25 to 90 mm or 25 to 50 mm (e.g., 35 mm) is coated with about 15 to 25 mg (e.g., 20 mg) of bone substitute material. The area (cm) of the layered cell sheet is ２ The amount of bone filler per 1000 mm is 1 to 4 g / cm ２ , 2 to 3 g / cm ２ , 2 g / cm ２In addition, the number of cells (1 × 10 ６ The amount of bone filler per 1×10 cells is 2 to 5 mg / 1×10 ６ cells, 2.5-4 mg / 1 x 10 ６ cells, 3mg / 1x10 ６ The cells can be selected from a variety of cells. Furthermore, by appropriately adjusting the number of cells at the time of seeding, the culture time, the culture temperature, and the like, a person skilled in the art can easily produce a layered cell sheet containing the desired number of cells. Regarding the determination of the number of osteogenic cells present on the surface of the composite, if a suitable cell number is confirmed in an experiment using rats (i.e., cells: rat osteogenic cells / transplantation target: rat), multiplying the suitable cell number by 0.67 can be considered to be the suitable cell number for humans.

[0027] 2. Method for Producing a Composite for Bone Regeneration or Augmentation The present invention also provides a method for producing a composite for bone regeneration or augmentation (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises the following steps: coating a bone substitute material with a stratified cell sheet containing osteogenic cells.

[0028] In one aspect, the manufacturing method of the present invention may further include a step of preparing a layered cell sheet containing osteogenic cells prior to the step of covering the bone filler with a layered cell sheet containing osteogenic cells.

[0029] The stratified cell sheet containing osteogenic cells in the production method of the present invention, the method for preparing the same, the bone filler, etc. are the same as those explained for the composite of the present invention.

[0030] In one embodiment, the manufacturing method of the present invention may further include a step of forming the composite into a roughly spherical shape, simultaneously with or after the step of coating the bone substitute with a stratified cell sheet containing osteogenic cells. The effects obtained by forming the composite into a roughly spherical shape and the method of forming the composite are also as described above. The preferred size of the roughly spherical composite and the number of osteogenic cells present on its surface are also as described above.

[0031] 3. Kit for Producing a Composite for Bone Regeneration or Augmentation The present invention also provides a kit for producing a composite for bone regeneration or augmentation (hereinafter, sometimes referred to as the "kit of the present invention"), which comprises a bone filler and a stratified cell sheet containing osteogenic cells.

[0032] The bone filler and the layered cell sheet containing osteogenic cells in the kit of the present invention are the same as those explained in the composite of the present invention.

[0033] The kit of the present invention may contain components other than the bone substitute material and the layered cell sheet containing osteogenic cells, such as, but not limited to, sterilized tweezers and instructions.

[0034] 4. Stratified cell sheet containing osteogenic cells for use in producing a composite for bone regeneration or augmentation The present invention also provides a stratified cell sheet containing osteogenic cells for use in producing a composite for bone regeneration or augmentation by combining it with a bone filler (hereinafter, sometimes referred to as the "stratified cell sheet of the present invention").

[0035] The layered cell sheet containing osteogenic cells, its preparation method, bone filler, etc. in the layered cell sheet of the present invention are the same as those explained for the composite of the present invention.

[0036] This embodiment can also be described as "a layered cell sheet composition for producing a composite for bone regeneration or augmentation, characterized in that the composite for bone regeneration or augmentation is produced by coating a bone filler with the layered cell sheet composition containing osteogenic cells." Alternatively, this embodiment can be described as "use of a layered cell sheet containing osteogenic cells for producing a composite for bone regeneration or augmentation, characterized in that the composite for bone regeneration or augmentation is produced by coating a bone filler with the layered cell sheet containing osteogenic cells."

[0037] 5. Bone Prosthetic Material for Use in Producing a Composite for Bone Regeneration or Augmentation The present invention also provides a bone prosthetic material (hereinafter sometimes referred to as the "bone prosthetic material of the present invention") for use in producing a composite for bone regeneration or augmentation by combining it with a stratified cell sheet containing osteogenic cells.

[0038] The bone filler, the stratified cell sheet containing osteogenic cells, and the preparation method thereof in the bone filler of the present invention are the same as those explained in the composite of the present invention.

[0039] This embodiment can also be described as "a bone filler composition for producing a composite for bone regeneration or augmentation, characterized in that the composite for bone regeneration or augmentation is produced by coating the bone filler with a stratified cell sheet containing osteogenic cells." Alternatively, this embodiment can be described as "use of a bone filler for producing a composite for bone regeneration or augmentation, characterized in that the composite for bone regeneration or augmentation is produced by coating the bone filler with a stratified cell sheet containing osteogenic cells."

[0040] 6. Method for Treating Bone Defect The present invention also provides a method for treating a bone defect in a subject (hereinafter referred to as the "therapeutic method of the present invention"), which comprises the step of transplanting the composite of the present invention into a subject having a bone defect.

[0041] In the treatment method of the present invention, the step of transplanting the composite of the present invention into a subject having a bone defect may be carried out by a method known per se. Briefly, a bone defect site or its vicinity (in other words, a site where bone regeneration or augmentation is intended) in a subject is exposed using a surgical technique, and the composite of the present invention is placed at or near the bone defect site using tweezers or the like, thereby transplanting the composite of the present invention into or near the bone defect site of the subject. Note that, upon transplantation, only one composite of the present invention may be placed at or near the bone defect site of the subject, or multiple composites may be placed as necessary.

[0042] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0043] [Example 1] Transplantation of the complex of the present invention prepared using rat femur-derived bone marrow mesenchymal stem cells into the parietal region of rats

[0044] [Cell culture of osteogenic cells and preparation of layered cell sheets] Bone marrow was collected from the femur of a 6-week-old male F344 rat (see Ando, ​​W. et al., Tissue Eng Part A. 2008;14(12):2041-9). The collected bone marrow was seeded in a 90 mm dish (Corning), and the proliferated cells were used as bone marrow mesenchymal stem cells (hereinafter sometimes referred to as "MSCs"). The culture medium used was α-MEM (Thermo Fisher Scientific) containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific). MSCs were cultured in 5% CO ２ The cells were cultured in a humidified incubator at 37°C. The medium was changed every three days. The cells were passaged using trypsin-EDTA (0.25%, Thermo Fisher Scientific) before they reached 100% confluence. At the second passage, 5.4 × 10 cells were cultured in a 35 mm dish. ６ The cells were seeded in a 35 mm dish at 100x the cell density. After seeding, α-MEM containing 10% FBS, 1% antibiotic-antimycotic, and 0.2 mM ascorbic acid was used as the medium. The medium was changed daily, and the cells were cultured for 14 days to prepare a layered cell sheet. The prepared layered cell sheet could be detached from the dish without any additional enzyme treatment or equipment. The layered cell sheet used was one in which the cells were layered, could be grasped with tweezers, and was strong enough to maintain an approximately spherical shape even when coated with bone filler.

[0045] [Preparation of the composite of the present invention] A composite of the present invention was prepared by covering 20 mg of β-TCP (OSPHERION (registered trademark), Olympus Corporation) as a bone filler with a stratified cell sheet. The composite of the present invention thus prepared was approximately spherical in shape with a diameter of approximately 4 mm. The prepared composite was implanted under the periosteum of the parietal bone of an F344 rat (the gray circular area in Figure 2(a)).

[0046] [Transplantation Experiment] Three 6-week-old male F344 rats (KBT Oriental, Ltd.) were used. The rats were anesthetized with inhalation anesthesia using isoflurane (1%-2%) and local anesthesia using xylocaine (0.2%, 0.1 ml). After anesthesia, the animals were cleaned with povidone-iodine, and an approximately 1 cm incision was made down to the periosteum, which was then peeled off to expose the parietal bone. Next, the composite of the present invention was transplanted under the periosteum, and the skin and periosteum were sutured with absorbable sutures. During surgery, xylocaine (2%, 0.1 ml) was administered locally to reduce postoperative pain. Furthermore, 6 mg / kg of gentamicin was administered intraperitoneally to prevent postoperative infection. After 12 weeks, the rats were euthanized, and the transplanted areas were harvested and subjected to HE staining. The results are shown in Figure 2(b).

[0047] As shown in Figure 2(b), vertical bone augmentation was achieved by implanting the composite of the present invention into the parietal region of a rat. Furthermore, new bone formation was confirmed not only from the host bone but also from the center of the composite of the present invention. Furthermore, while applying a bone substitute to a transplant site is generally a relatively difficult process due to the small particle size of the bone substitute, the use of the composite of the present invention made the transplantation process extremely easy.

[0048] [Example 2] Transplantation of the complex of the present invention prepared using human iliac bone marrow mesenchymal stem cells into immunodeficient mice

[0049] [Cells] Three strains of human iliac bone marrow mesenchymal stem cells (MSCs) were purchased from Lonza. The culture medium used was α-MEM (Thermo Fisher Scientific) containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific). MSCs were seeded in a 90 mm dish and incubated at 5% CO ２ The cells were cultured in a humidified incubator at 37°C. The medium was changed every three days. Cells were passaged using trypsin-EDTA (0.25%, Thermo Fisher Scientific) before they reached 100% confluence. At the 3rd to 6th passages, 3.6 x 10 cells were cultured in a 35 mm dish. ６The cells were seeded at 100 cells / dish. After seeding on a 35 mm dish, the medium used was α-MEM containing 10% FBS, 1% antibiotic-antimycotic, and 0.2 mM ascorbic acid. The medium was changed daily, and the cells were cultured for 7 days to prepare a layered cell sheet.

[0050] [Preparation of the Composite of the Present Invention] 50 mg of carbonate apatite (Cytrans® Granules, Fordi Co., Ltd.) was coated with a layered cell sheet of each of the three strains to prepare the composite of the present invention. As a control, β-TCP was mixed with 3% atelocollagen (Koken Co., Ltd.) and incubated at 37°C for 30 minutes for gelation to prepare a composite. These four types of composites were implanted under the periosteum of the parietal bone of C.B-17 SCID mice.

[0051] [Transplantation Experiment] Twelve 4-week-old male C.B-17 SCID mice (KBT Oriental Co., Ltd.) were used. Mice were anesthetized with inhalation anesthesia using isoflurane (1%-2%) and local anesthesia using xylocaine (0.2%, 0.1 ml). After anesthesia, the animals were cleaned with povidone-iodine, and an approximately 1 cm incision was made down to the periosteum, which was then peeled off to expose the parietal bone. Next, four types of complexes were transplanted under the periosteum of three mice each, and the skin and periosteum were sutured with absorbable sutures. During surgery, xylocaine (2%, 0.1 ml) was administered locally to reduce postoperative pain. Furthermore, 6 mg / kg of gentamicin was administered intraperitoneally to prevent postoperative infection. After 12 weeks, the mice were euthanized, and the transplanted areas were harvested and subjected to HE staining and human-specific vimentin immunostaining. The results are shown in Figure 3.

[0052] As shown in Figure 3(a), bone growth was observed in the composite of the present invention prepared using human-derived mesenchymal stem cells. Furthermore, immunostaining for human-specific vimentin demonstrated that cells present on the surface of the composite of the present invention are directly involved in bone formation, and that some of these cells migrated to the center of the composite after implantation. It has been reported that implanted cells do not remain in the localized area and become undetectable over time (Zimmermann, CE et al., Tissue Eng Part A. 2011;17(7-9):1147-56). However, in this example, the implanted cells not only survived but also differentiated into osteocytes 12 weeks after implantation. This is thought to be due to the maintenance of a cell-surviving environment by the layered cells and their derived extracellular matrix, and the paracrine and autocrine effects of cytokines secreted by the surviving cells. Furthermore, as in Example 1, new bone formation was observed not only from the host bone but also from the center of the composite of the present invention. Furthermore, compared with the control, the rate of new bone formation in the complex of the present invention was increased by more than two times, demonstrating that the complex of the present invention promotes new bone formation extremely efficiently (Figure 3(b)).

[0053] [Example 3] Examination of the size of the complex of the present invention

[0054] [Cells] Bone marrow was collected from the femur of a 6-week-old male F344 rat. The collected bone marrow was seeded in a 90 mm dish (Corning), and the expanded cells were used as MSCs. The culture medium used was α-MEM (Thermo Fisher Scientific) containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific). MSCs were cultured in 5% CO ２ The cells were cultured in a humidified incubator at 37°C with the medium changed every three days. The cells were passaged using trypsin-EDTA (0.25%, Thermo Fisher Scientific) before they reached 100% confluence. For the second passage, the cells were seeded into 35 mm, 60 mm, or 90 mm dishes. 5.4 × 10 cells were seeded into 35 mm dishes.６ cells / dish, 60 mm dish is 11.0 x 10 ６ cells / dish, 90mm dish is 32.0 x 10 ６ The cells were seeded at 1000 cells / dish. After seeding, the medium used for each dish was α-MEM containing 10% FBS, 1% antibiotic-antimycotic, and 0.2 mM ascorbic acid. The medium was changed daily, and the cells were cultured for 14 days to prepare a layered cell sheet.

[0055] [Preparation of the Composite of the Present Invention] Composites of the present invention of different sizes were prepared by covering 20 mg of β-TCP (OSFERION®) in a 35 mm dish, 40 mg in a 60 mm dish, and 120 mg in a 90 mm dish with a layered cell sheet. The ratio of cell number to culture area and bone filler weight was the same. The dishes, dish area, cell number, and bone filler amount used are shown in Table 1. The appearances of the composites of the present invention prepared in different sizes are shown in Figure 4. The composite prepared in the 35 mm dish was approximately spherical with major and minor axes both approximately 4 mm, the composite prepared in the 60 mm dish was approximately spherical with major and minor axes both approximately 6 mm, and the composite prepared in the 90 mm dish was approximately spherical with a major axis of approximately 10 mm and a minor axis of approximately 6 mm. These composites were implanted under the periosteum of the parietal bone of F344 rats.

[0056]

[0057] [Transplantation Experiment] Three 6-week-old male F344 rats (KBT Oriental Co., Ltd.) were used. The rats were anesthetized with inhalation anesthesia using isoflurane (1%-2%) and local anesthesia using xylocaine (0.2%, 0.1 ml). After anesthesia, the animals were cleaned with povidone-iodine, and an approximately 1 cm incision was made down to the periosteum, which was then peeled off to expose the parietal bone. The composites prepared in each dish were then transplanted under the periosteum (N=1), and the skin and periosteum were sutured with absorbable sutures. During surgery, xylocaine (2%, 0.1 ml) was administered locally to reduce postoperative pain. Additionally, 6 mg / kg of gentamicin was administered intraperitoneally to prevent postoperative infection. After 12 weeks, the rats were euthanized, and the transplanted areas were harvested and subjected to HE staining. The results are shown in Figure 5.

[0058] As shown in Figure 5, good bone augmentation was confirmed even with the composite of the present invention having a larger size. Even when a large-sized composite of the present invention was implanted, bone formation was confirmed on the host bone side, the center of the composite, and the surface side. In rats implanted with the composite of the present invention prepared in a 90 mm dish, the formation of bone marrow-like tissue was confirmed (the two arrows in Figure 5). The larger the size of the composite, the easier it was to achieve greater bone augmentation.

[0059] [Example 4] Transplantation of the complexes of the present invention of different sizes under the condition of uniform cell number

[0060] [Cells] Bone marrow was collected from the femur of a 6-week-old male F344 rat. The collected bone marrow was seeded onto a 90 mm dish (Corning). The expanded cells were used as MSCs. The culture medium used was α-MEM (Thermo Fisher Scientific) containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific). MSCs were cultured in 5% CO ２ The cells were cultured in a humidified incubator at 37°C with the medium changed every three days. The cells were passaged using trypsin-EDTA (0.25%, Thermo Fisher Scientific) before they reached 100% confluence. At the second passage, the cells were subcultured into 35 mm or 90 mm dishes. The 35 mm dishes contained 5.4 × 10 ６ cells / dish, 90mm dish is 32.0 x 10 ６ The cells were passaged to form 100 cells / dish. After seeding in each dish, α-MEM containing 10% FBS, 1% antibiotic-antimycotic, and 0.2 mM ascorbic acid was used as the medium. The medium was changed daily, and the cells were cultured for 14 days to prepare a layered cell sheet.

[0061] [Preparation of the complex of the present invention] 20 mg of β-TCP (Ospherion®) was coated on a 35 mm dish, and 120 mg of β-TCP (Ospherion®) was coated on a 90 mm dish with a layered cell sheet to prepare the complex of the present invention. These complexes were implanted under the periosteum of the parietal bone of F344 rats.

[0062] [Transplantation Experiment] Four 6-week-old male F344 rats (KBT Oriental Co., Ltd.) were used. The rats were anesthetized with inhalation anesthesia using isoflurane (1%-2%) and local anesthesia using xylocaine (0.2%, 0.1 ml). After anesthesia, the animals were cleaned with povidone-iodine, and an approximately 1 cm incision was made down to the periosteum, which was then peeled off to expose the parietal bone. The composites prepared in each dish were then transplanted under the periosteum. One composite prepared in a 90 mm dish was transplanted per rat, and six composites prepared in a 35 mm dish were transplanted per rat, for a total of three rats. The diameter of the dish used, the number of composites of the present invention transplanted, and the total number of cells transplanted per rat are shown in Table 2. After transplantation, the skin and periosteum were sutured with absorbable sutures. During surgery, xylocaine (2%, 0.1 ml) was administered locally to alleviate postoperative pain. Furthermore, 6 mg / kg of gentamicin was administered intraperitoneally to prevent postoperative infection. After 12 weeks, the rats were euthanized, and the transplanted sites were harvested and subjected to HE staining. The results are shown in Figure 6.

[0063]

[0064] As shown in Figure 6, good bone augmentation was confirmed for all sizes of composites of the present invention. When six composites of the present invention prepared in a 35 mm dish were implanted, more favorable vertical bone augmentation was achieved compared to when one composite of the present invention prepared in a 90 mm dish was implanted. Furthermore, the composites of the present invention prepared in a 35 mm dish were preferable to the composites of the present invention prepared in a 90 mm dish in terms of ease of handling.

[0065] [Example 5] Investigation of the number of cells and number of days of culture required for preparation of stratified cell sheets

[0066] Bone marrow was collected from the femur of 6-week-old male F344 rats. The collected bone marrow was seeded onto a 90 mm dish (Corning). The expanded cells were used as MSCs. The culture medium used was α-MEM (Thermo Fisher Scientific) containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific). MSCs were cultured in 5% CO ２ The cells were cultured in a humidified incubator at 37°C with the medium changed every three days. The cells were passaged using trypsin-EDTA (0.25%, Thermo Fisher Scientific) before they reached 100% confluence. For the second passage, 1.8 × 10 cells were cultured in a 35 mm dish. ６ cells / dish, 3.6×10 ６ cells / dish, 5.4×10 ６ cells / dish, or 7.2 x 10 ６ The cells were passaged to form cells / dish, and the medium was changed daily. The cells were cultured for a maximum of 14 days, and the number of culture days required for preparation of a layered cell sheet was investigated. The results are shown in Table 3. The symbols in the table mean the following: ×: A layered cell sheet could not be prepared. △: A layered cell sheet could be prepared. ○: A good layered cell sheet could be prepared.

[0067]

[0068] As shown in Table 3, the number of days of culture required for sheet preparation varies depending on the number of cells before culture. ６ It was shown that if approximately 100 mesenchymal stem cells can be collected, it is possible to prepare a good stratified cell sheet (and the complex of the present invention) within two weeks.

[0069] According to the present invention, it is possible to treat a wide range of bone defects requiring horizontal / vertical bone regeneration and / or bone augmentation. Furthermore, according to the present invention, it is possible to efficiently prepare a novel composite for bone regeneration or augmentation that enables such treatment. Therefore, the present invention is extremely useful in the fields of orthopedics and dentistry.

[0070] This application is based on patent application No. 2023-102016 filed in Japan (filing date: June 21, 2023), the contents of which are incorporated in full herein. In addition, all documents cited in this specification are incorporated by reference in their entirety into this application.

Claims

1. A bone regeneration or proliferation complex (excluding those using a combination of two or more cell sheets) comprising a bone graft material covered with a layered cell sheet containing osteogenic cells, wherein the layered cell sheet is 9.5 cm ２ 3 x 10 ６ ~10 x 10 ６ A complex containing cells.

2. The composite according to claim 1, wherein the bone graft material is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

3. The complex according to claim 1 or 2, wherein the osteogenic cell is at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

4. A method for producing a bone regeneration or proliferation complex (excluding those using a combination of two or more cell sheets), comprising the following steps: The process involves covering the bone graft material with a layered cell sheet containing osteogenic cells, where the layered cell sheet is 9.5 cm ２ 3 x 10 ６ ~10 x 10 ６ A method involving cells.

5. A method for producing a bone regeneration or proliferation complex according to claim 4, comprising the step of preparing a layered cell sheet containing osteogenic cells before the step of covering the bone graft material with a layered cell sheet containing osteogenic cells.

6. The manufacturing method according to claim 4 or 5, wherein the bone graft material is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

7. The method for producing a bone marrow cell according to claim 4 or 5, wherein the osteogenic cell is at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

8. A kit for producing a bone regeneration or proliferation composite (excluding those using two or more combined cell sheets) comprising a bone filler and a layered cell sheet containing osteogenic cells, wherein the layered cell sheet contains 3×10 ２ per 9.5 cm ６ to 10×10 ６ cells, the kit.

9. The kit according to claim 8, wherein the bone graft material is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

10. The kit according to claim 8 or 9, wherein the osteogenic cell is at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

11. A stratified cell sheet containing osteogenic cells, for use in manufacturing a bone regeneration or proliferation complex (excluding those using a combination of two or more cell sheets) by combining it with a bone graft material, wherein the stratified cell sheet is 9.5 cm ２ 3 x 10 ６ ×10×10 ６ A sheet containing cells.

12. A layered cell sheet containing osteogenic cells according to claim 11, wherein the bone graft material is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

13. The stratified cell sheet according to claim 11 or 12, wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.

14. A bone graft material for use in manufacturing a bone regeneration or proliferation complex (excluding those using a combination of two or more cell sheets) by combining it with a layered cell sheet containing osteogenic cells, wherein the layered cell sheet is 9.5 cm ２ 3 x 10 ６ ~10 x 10 ６ A bone graft material containing cells.

15. The bone graft material according to claim 14, wherein the bone graft material is at least one selected from the group consisting of hydroxyapatite, β-tricalcium phosphate, and carbonate apatite.

16. The bone graft material according to claim 14 or 15, wherein the osteogenic cells are at least one selected from the group consisting of mesenchymal stem cells, osteoblasts, and bone marrow-derived stromal cells.