Bone-like tissue and method for producing same

Abstract

The method that produces bone-like tissue according to the present disclosure cultures a three-dimensional mesenchymal stem cell aggregate embedded in a gel using bone differentiation induction medium. This method does not use an artificial scaffold material.

Description

[Technical field]

[0001] The present invention relates to a bone-like tissue and a method for producing the same, and more particularly to a bone-like tissue that does not contain artificial materials and a method for producing the same. [Background technology]

[0002] Conventionally, autologous bone grafting, in which bone is isolated from the chin or iliac bone and transplanted, has been performed as a bone regeneration therapy for patients with extensive bone defects after osteotomy due to severe fractures, periodontitis, or tumors. This is an ideal bone regeneration therapy because it supplies bone cells and bone matrix to the bone defect area, but there is a limit to the amount that can be harvested, and in reality, there are many cases where treatment is difficult due to a lack of bone tissue.

[0003] On the other hand, mesenchymal stem cells (MSCs), which have multipotency and self-proliferation ability, are considered to be suitable cells for tissue regeneration therapy, and in recent years, many attempts have been made to produce and supply three-dimensional bone-like tissue equivalent to autologous bone by mixing MSCs with artificial scaffold materials. For example, it is known that a three-dimensional graft with high bone regeneration ability can be obtained by mixing MSCs with hydroxyapatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel, etc., and culturing the mixture in a bone differentiation induction medium (medium containing dexamethasone, ascorbic acid, and β-glycerophosphate) established in a two-dimensional culture experimental system (see Non-Patent Document 1).

[0004] However, none of the above methods have been able to create three-dimensional bone-like tissue similar to bone, and no bone regeneration effect equivalent to that of autologous bone grafts has been achieved to date. The reason for this is thought to be that artificial scaffold materials do not become bone matrix produced by cells, but are merely foreign bodies to the body. In addition, as long as artificial materials are used as scaffolds, it is impossible to induce MSCs to become bone cells that are contained within the bone matrix and form a network, no matter how much they are cultured in bone differentiation medium. In other words, in order to create three-dimensional bone-like tissue equivalent to autologous bone that can be applied as a more effective bone regeneration therapy, a new culture method is needed that does not use artificial materials, but rather cultures MSCs three-dimensionally, produces bone matrix, and induces them to become bone cells contained within it.

[0005] To date, attempts have been made to create three-dimensional bone-like tissue by culturing MSCs spheroids, which are primarily based on cell-cell adhesion, and three-dimensional cell aggregates (C-MSCs) constructed from the extracellular matrix (ECM) produced by MSCs, in osteogenic differentiation medium (dexamethasone (100 nM) + β-glycerophosphate (10 mM) + ascorbic acid (50 μg / ml)) as representative three-dimensional culture methods for MSCs that do not use artificial materials. However, although mineral deposition can be induced in both MSCs spheroids and C-MSCs, appropriate bone matrix formation and induction of bone cells could not be achieved (see Non-Patent Documents 2 and 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Amini AR et al., Crit Rev Biomed Eng, 2012, 40(5), 363-408. [Non-Patent Document 2] Langenbach F, Clin Oral Invest, 2013, 17(1), 9-17. [Non-Patent Document 3] Kittaka M et al., Cytotherapy, 2015, 17(7), 860-873. Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, the combination of artificial scaffold materials and MSCs is attracting much attention in the field of bone regeneration therapy, but the technology to create three-dimensional bone-like tissue equivalent to bone has not yet been developed. In order to create and supply three-dimensional bone-like tissue applicable to effective bone regeneration therapy, it is necessary to achieve bone tissue formation while culturing MSCs three-dimensionally without using artificial scaffold materials.

[0008] The present invention has been made in consideration of the above-mentioned problems, and its object is to enable bone tissue formation while culturing MSCs in three dimensions without using an artificial scaffold material, thereby obtaining three-dimensional bone-like tissue that is effective for bone regeneration therapy. [Means for solving the problem]

[0009] In order to achieve the above-mentioned object, the inventors conducted extensive research and discovered that by embedding a three-dimensional mesenchymal stem cell cluster in a gel and culturing it in a bone differentiation-inducing medium, it is possible to produce a three-dimensional bone-like tissue consisting of a bone matrix onto which minerals are deposited and bone cells contained within the bone matrix without using an artificial scaffold material, thereby completing the present invention.

[0010] Specifically, the method for producing bone-like tissue according to the present invention comprises a step of culturing a three-dimensional mesenchymal stem cell cluster embedded in a gel using a bone differentiation induction medium, and is characterized by not using an artificial scaffold material.

[0011] According to the method for producing bone-like tissue of the present invention, it is possible to easily obtain bone-like tissue suitable for transplantation into a bone defect, which contains a bone matrix on which minerals are deposited and bone cells contained within the bone matrix, and does not contain foreign substances such as artificial scaffold materials.

[0012] It is preferable that the method for producing bone-like tissue according to the present invention further comprises the steps of adhesively culturing mesenchymal stem cells in a culture vessel to obtain a cell sheet, detaching the cell sheet from the culture vessel, and performing suspension culture on the detached cell sheet to obtain a three-dimensional mesenchymal stem cell cluster.

[0013] In the method for producing bone-like tissue according to the present invention, the gel is preferably a gel containing polysaccharide as a main component.

[0014] In addition, in the method for producing bone-like tissue according to the present invention, it is preferable that the gel does not contain any extracellular matrix components other than those generated from the embedded cells.

[0015] In the method for producing bone-like tissue according to the present invention, the bone differentiation-inducing medium preferably contains ascorbic acid and β-glycerophosphate.

[0016] In the method for producing bone-like tissue according to the present invention, the bone differentiation-inducing medium preferably contains dexamethasone.

[0017] On the other hand, the bone-like tissue according to the present invention is characterized in that it contains a bone matrix on which minerals are deposited and bone cells contained in the bone matrix, does not contain an artificial scaffold material, and the bone cells form a network. Also, it is preferable that the bone-like tissue according to the present invention does not contain blood vessels.

[0018] The bone-like tissue of the present invention contains a bone matrix and bone cells contained within the bone matrix, but does not contain a foreign artificial scaffold material, and is therefore suitable for transplantation into bone defects and can be effectively used in bone regeneration therapy.

[0019] The pharmaceutical composition according to the present invention is characterized in that it is a pharmaceutical composition for bone regeneration, which contains the bone-like tissue according to the present invention.

[0020] Since the pharmaceutical composition according to the present invention contains the above-mentioned bone-like tissue, it is suitable for transplantation into bone defects and can be effectively used in bone regeneration therapy. Effect of the Invention

[0021] According to the bone-like tissue and the method for producing the same of the present invention, it is possible to obtain three-dimensional bone-like tissue that is effective for bone regeneration therapy and contains a bone matrix on which minerals are deposited and bone cells contained within the bone matrix. [Brief description of the drawings]

[0022] [Figure 1] 1 shows photographs showing the results of Alizarin Red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in gel and culturing them in a concentration-optimized bone differentiation-inducing medium. [Diagram 2] 1 shows photographs depicting the results of alizarin red staining or HE staining of paraffin-embedded sections of cell clusters obtained by suspension culture of C-MSCs using a conventional osteogenic differentiation-inducing medium. [Diagram 3] 1 shows photographs depicting the results of alizarin red staining or HE staining of paraffin-embedded sections of cell clusters obtained by suspension culture of C-MSCs in a concentration-optimized osteodifferentiation-inducing medium. [Figure 4] 1 shows photographs showing the results of Alizarin Red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in gel and culturing them in a conventional bone differentiation-inducing medium. [Diagram 5] This is a photograph obtained by micro-CT imaging of three-dimensional bone-like tissue obtained by embedding C-MSCs in gel and culturing them in an optimized concentration of bone differentiation-inducing medium. [Figure 6] (a) to (c) are photographs showing the results of immunostaining for bone matrix proteins on paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in gel and culturing them in a concentration-optimized bone differentiation-inducing medium, where (a) shows the results of staining for COL1, (b) shows the results of staining for OPN, and (c) shows the results of staining for OCN. [Figure 7]Fig. 11 is a photograph showing the results of F-actin immunostaining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in gel and culturing them in an optimized concentration of bone differentiation-inducing medium. [Figure 8] (a) to (c) are photographs showing the condition of the transplant site 4 weeks after subcutaneous transplantation of three-dimensional bone-like tissue into a SCID mouse. (a) is a photograph showing the results of micro-CT imaging of the subcutaneous transplant site in the mouse. (b) is a photograph showing the results of HE staining of a section prepared after removing the subcutaneous transplant site from the mouse together with the three-dimensional bone-like tissue and decalcifying it. (c) is a photograph showing the results of AZN staining of the same section. [Figure 9] Photographs (a) to (c) show the condition of the transplant site 8 weeks after subcutaneous transplantation of three-dimensional bone-like tissue into a SCID mouse. Photograph (a) shows the results of micro-CT scanning of the subcutaneous transplant site in the mouse. Photograph (b) shows the results of HE staining of a section prepared after removing the subcutaneous transplant site from the mouse together with the three-dimensional bone-like tissue and decalcifying it. Photograph (c) shows the results of AZN staining of the same section. [Figure 10] 1 is a photograph showing the state in which a three-dimensional bone-like tissue was transplanted into a bone defect in the calvarium of a nude rat. [Figure 11] 1 is a HE stained photograph showing a calvarial bone defect in a SCID mouse, showing the result 4 weeks after the defect was created when no three-dimensional bone-like tissue was transplanted into the defect. [Figure 12] 1 is a HE stained photograph showing a calvarial bone defect in a SCID mouse, showing the result 4 weeks after transplanting a three-dimensional bone-like tissue into the bone defect. [Figure 13] (a) is an HE-stained photograph showing the result of transplanting three-dimensional bone-like tissue into a calvarial bone defect in a nude rat 8 weeks after the transplantation, and (b) and (c) are enlarged views of the areas enclosed in the squares in (a). [Figure 14] (a) is an AZAN-stained photograph showing the result of transplanting three-dimensional bone-like tissue into a calvarial bone defect in a nude rat 8 weeks after the transplantation, and (b) and (c) are enlarged views of the areas enclosed in the boxes in (a). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following preferred embodiments are merely exemplary in nature and are not intended to limit the present invention, its application method, or its uses.

[0024] The bone-like tissue according to the present invention is characterized in that it contains a bone matrix on which minerals are deposited and bone cells contained in the bone matrix, does not contain an artificial scaffold material, and the bone cells form a network. In addition, the bone-like tissue according to the present invention preferably does not contain blood vessels.

[0025] In the bone-like tissue according to the embodiment of the present invention, the bone matrix is ​​an accumulation of substances produced by cells having bone formation ability and secreted outside the cells, and includes, but is not limited to, calcium phosphate and the like, including proteins such as osteocalcin, osteopontin, osteonectin, and collagen. In addition, bone cells are the cells that are most abundant in bone, and are encapsulated in the bone matrix to form a network with each other and constitute bone together with the bone matrix. Here, the formation of a network refers to the formation of a state in which bone cells are connected to each other three-dimensionally. In particular, this refers to a state in which proteins related to the cytoskeleton, such as F-actin, are expressed in bone cells, and the cells extend their processes to connect with each other to form a three-dimensional network.

[0026] In this embodiment, the term "minerals" specifically includes calcium, magnesium, and phosphorus. In this embodiment, the term "mineral deposition" refers to a state in which at least calcium is deposited in the bone matrix, and in this case, magnesium or phosphorus may also be deposited. To confirm the presence or absence of mineral deposition, for example, a method of staining a tissue section using alizarin red, which is a dye that binds to calcium, can be used, but the present invention is not limited to this, and other methods can also be used as long as they can confirm the presence of minerals in the bone matrix.

[0027] In the bone-like tissue of this embodiment, the artificial scaffold material is an artificial scaffold material that has conventionally been used for three-dimensional cell culture, and examples thereof include, but are not limited to, hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen gel, and hyaluronic acid gel.

[0028] The bone-like tissue according to the present embodiment does not contain the above-mentioned artificial scaffold material, i.e., does not contain any foreign matter for bone tissue, and therefore has a good network of bone cells formed in the bone matrix, making it an extremely excellent transplant material for bone regeneration therapy. In addition, in the bone-like tissue according to the present embodiment, the bone cells are preferably derived from mesenchymal stem cells artificially cultured. In addition, the bone-like tissue according to the present embodiment is preferably produced by the method for producing bone-like tissue according to the present invention. In addition, the bone-like tissue according to the present embodiment is preferably produced in an ex vivo culture system by the method for producing bone-like tissue according to the present invention, and therefore does not contain blood vessels in the tissue.

[0029] The method for producing bone-like tissue according to the present invention is a method for producing three-dimensional bone-like tissue that does not contain artificial scaffold materials by culturing three-dimensional mesenchymal stem cell clusters embedded in a gel using a bone differentiation induction medium without using artificial scaffold materials.

[0030] In the method for producing bone-like tissue according to one embodiment of the present invention, the artificial scaffold material is an artificial scaffold material that has been conventionally used in three-dimensional cell culture, as described above, and examples thereof include, but are not limited to, hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel, etc. In addition, in the method for producing bone-like tissue according to this embodiment, the mesenchymal stem cells include, but are not limited to, those collected from tissues such as marrow, adipose tissue, placental tissue, umbilical cord tissue, dental pulp, etc. of animals such as humans, as well as mesenchymal stem cells derived from induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0031] In addition, in the method for producing bone-like tissue according to the present embodiment, the three-dimensional mesenchymal stem cell clump is a cell clump composed of a plurality of mesenchymal stem cells, and is not a two-dimensional flat sheet-like cell clump, but a three-dimensional granular cell clump. Normally, when mesenchymal stem cells are cultured by adhesion, they grow two-dimensionally, that is, in a sheet-like form to form a cell sheet. In the present embodiment, it is preferable to culture the mesenchymal stem cells by adhesion in a culture vessel before embedding them in a gel and culturing them to form a sheet (cell sheet), and then detach the cell sheet from the culture vessel, and then culture the cell sheet in suspension to obtain a three-dimensional mesenchymal stem cell clump, and then culture the cell clump in a state embedded in a gel using an osteogenic differentiation-inducing medium. The growth medium used to obtain this cell mass is not particularly limited as long as it is a medium capable of growing mesenchymal stem cells and contains factors that induce the production of extracellular matrix such as ascorbic acid. For example, High glucose DMEM (Sigma) containing FBS and a specified antibiotic in addition to the above factors can be used. In addition, Prime-XV MSC expansion XSFM (Irvine) which does not contain xenogeneic animal proteins can be used.

[0032] In the method for producing bone-like tissue according to the present embodiment, the gel is not particularly limited in terms of its constituents, so long as it can be cultured by embedding a three-dimensional mesenchymal stem cell cluster inside, but it is preferable that the gel is mainly composed of polysaccharides, and for example, Vitrogel (manufactured by The Well Biosciences) can be used. The hardness of the gel can be adjusted as appropriate. In the case of Vitrogel, the hardness can be adjusted by diluting the concentration when solidifying the gel. For example, a gel made with Vitrogel:PBS=1:1 is hard, and a gel made with 1:5 is soft and semi-solid. For the induction of three-dimensional bone-like tissue, it is preferable to use Vitrogel:PBS=1:3. In addition to gels mainly composed of polysaccharides, gels mainly composed of extracellular matrix (ECM) such as collagen and laminin can also be used, but since they contain a large amount of cell adhesion factors, cells will migrate from the cell clusters, and the resulting three-dimensional bone-like tissue will become smaller, so it is preferable that the gel used does not contain extracellular matrix components. Therefore, even during the culture process, it is preferable that the gel does not contain any extracellular matrix components other than those generated by the embedded cells.

[0033] In the method for producing bone-like tissue according to the present embodiment, the bone differentiation-inducing medium may be any of various well-known bone differentiation-inducing media, but preferably contains ascorbic acid and β-glycerophosphate, and more preferably contains dexamethasone. Instead of dexamethasone, recombinant proteins of BMP2 or Wnt3a may be contained. MSCgo Osteogenic differentiation medium (Biological Industries), which does not contain xenogeneic animal proteins, may also be used.

[0034] The pharmaceutical composition according to the present invention is a pharmaceutical composition containing the bone-like tissue according to the present invention and used for bone regeneration.

[0035] The pharmaceutical composition according to one embodiment of the present invention is used for the purpose of bone regeneration, and is applicable to any bone without any limitation. It is preferably applicable to bone regeneration therapy for intractable fractures, periodontitis, or cases of extensive bone loss after osteotomy, although it is not particularly limited to the following.

[0036] The pharmaceutical composition according to the present embodiment may contain various pharma- ceutical acceptable additives in addition to the bone-like tissue according to the present invention. The pharmaceutical composition according to the present embodiment is not particularly limited in its form, but is preferably in a powder or granular form so that it can be directly transplanted into bone defects of various shapes. EXAMPLES

[0037] Examples are given below to explain in detail the bone-like tissue and the method for producing the same according to the present invention.

[0038] [Example 1: Cultivation of mesenchymal stem cells and preparation of three-dimensional bone-like tissue] (Preparation of C-MSCs) Human bone marrow-derived mesenchymal stem cells (MSCs, obtained from RIKEN) were seeded in a 96-well plate (Corning) at a cell density of 5.0×104 cells / well, and cultured for 4 days in a growth medium (GM medium) consisting of High glucose DMEM (Sigma) containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, plus 50 μg / ml ascorbic acid (Sigma), to produce sufficient ECM. After that, the cells were attached to the 96-well plate, and the sheet-like cell population (cell sheet) was bluntly peeled off. Specifically, the cell sheet can be isolated from the plate by moving a thin rod around the inner wall of the well while applying it to the edge of the cell sheet attached to the plate. In this way, the cell sheet composed of ECM and MSCs was suspended. The resulting MSCs / ECM complexes were transferred to an ultra-low binding plate (Iwaki) and cultured for 3 days in osteogenic differentiation-inducing medium (GM medium supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg / ml ascorbic acid). As a result, C-MSCs, which are spherical cell clusters with diameters of approximately 600 μm to 800 μm, were obtained.

[0039] (Induction of three-dimensional bone-like tissue from C-MSCs) The C-MSCs obtained as described above were embedded in a hydrogel mainly composed of polysaccharide (Vitrogel 3D-RGD (500 μl / 48 well): manufactured by The Well Biosciences), and 250 μl of bone differentiation induction medium (concentration-optimized bone differentiation induction medium) containing 10 nM dexamethasone, 5 mM β-glycerophosphate, and 50 μg / ml ascorbic acid added to the GM medium in a 48-well plate was added and cultured for 14 days (Example). Specifically, the gel containing C-MSCs was placed in the well, and then the medium was added on top of the gel. As a result, a three-dimensional bone-like tissue that was a roughly spherical granule with a diameter of about 1 mm was obtained. On the other hand, as a comparative example, C-MSCs were cultured for 14 days in a floating state using a bone differentiation induction medium (Comparative Example 1) or a concentration-optimized bone differentiation induction medium (Comparative Example 2) without being embedded in the gel. As a result, spherical calcified material was obtained in Comparative Example 1, and no significant change from the spherical cell aggregate was observed in Comparative Example 2. In Comparative Example 3, C-MSCs were embedded in gel as described above and cultured in a conventional bone differentiation-inducing medium. The histological evaluation of the three-dimensional bone-like tissue or cell aggregate obtained in each Example and Comparative Example is described below.

[0040] (Histological evaluation of three-dimensional bone-like tissue) In order to observe the degree of calcification in the three-dimensional bone-like tissues or cell clusters obtained by the above-mentioned cultures in the Examples and Comparative Examples, they were fixed with 1% formaldehyde, embedded in paraffin, and then 5 μm sections were prepared and stained with Alizarin Red (except Comparative Example 3). In order to observe their histological structures, the obtained samples were decalcified with 10% ethylenediaminetetraacetic acid, and then 5 μm sections were prepared and stained with HE. The results are shown in Figures 1 to 4. In addition, the results of CT imaging of the three-dimensional bone-like tissues of the above-mentioned Examples are shown in Figure 5. Furthermore, the sections of the three-dimensional bone-like tissues of the above-mentioned Examples were immunostained for bone matrix proteins COL1, OPN, and OCN, and for cytoskeleton F-actin, using standard methods, and the results of observation with a confocal laser microscope are shown in Figures 6(a) to (c) and Figure 7, respectively.

[0041] As shown in FIG. 1, in the Example in which C-MSCs were embedded in gel and cultured in a concentration-optimized bone differentiation-inducing medium, abundant bone matrix and minerals deposited in the bone matrix stained with alizarin red were observed, and in the HE staining image, bone-like matrix stained with HE, osteoblast-like cells surrounding it, and osteocyte-like cells inside were observed. That is, in the Example, it was confirmed that a cell mass consisting of bone matrix-like tissue containing osteocyte-like cells, that is, a three-dimensional bone-like tissue, was obtained. In contrast, in Comparative Example 1, as shown in FIG. 2, deposition of minerals stained with alizarin red was observed, but when decalcified sections were made and observed histologically, no bone matrix structure was observed, and many of the cells were in a state of cell death indicated by nuclear shrinkage. The cause of cell death is thought to be excessive calcification effect by the added factor used for bone differentiation induction. On the other hand, in Comparative Example 2, in which a concentration-optimized bone differentiation-inducing medium containing low concentrations of dexamethasone and β-glycerophosphate was used, as shown in FIG. 3, cell death was reduced, but no mineral deposition into the ECM was obtained.

[0042] From the above, it was revealed that good three-dimensional bone-like tissue was obtained in the Example in which C-MSCs were embedded in gel and cultured in a concentration-optimized bone differentiation induction medium in which the concentration of the additive factor used for bone differentiation induction was reduced, but three-dimensional bone-like tissue was not obtained in Comparative Examples 1 and 2 in which suspension culture was performed without gel-embedded culture. In other words, it is suggested that in order to obtain good three-dimensional bone-like tissue, it is necessary to embed C-MSCs in gel and culture them.

[0043] Furthermore, when gel-embedded culture was performed while Comparative Example 3, in which a conventional bone differentiation-inducing medium was used, was prepared as a decalcified section and observed histologically, many cells were in a state of cell death as indicated by nuclear shrinkage, and the amount of bone matrix was also low, as compared with the Examples, as shown in Figure 4. Therefore, in order to obtain good three-dimensional bone-like tissue, it is considered preferable to reduce and optimize the concentration of additive factors used for bone differentiation induction in addition to gel-embedded culture of C-MSCs.

[0044] In addition, the three-dimensional bone-like tissue according to the present embodiment showed X-ray opacity as a whole, similar to human bone tissue, as shown in FIG. 5. Furthermore, in the three-dimensional bone-like tissue according to the present embodiment, as shown in FIG. 6(a)-(c), expression of COL1, OPN, and OCN was observed in the area stained with HE shown in FIG. 1 (the white area inside the three-dimensional bone-like tissue in FIG. 6(a)-(c)). Furthermore, as shown in FIG. 7, in the three-dimensional bone-like tissue according to the present embodiment, F-actin was expressed overall in the cells, and each cell formed a network while projecting projections, showing characteristics of bone cells. From the above results, it is considered that a three-dimensional bone-like tissue having characteristics equivalent to those of human bone tissue was obtained in the present embodiment.

[0045] [Example 2: Evaluation of autonomous bone formation ability of three-dimensional bone-like tissue] Next, the autonomous bone formation ability of the three-dimensional bone-like tissue obtained as described above was evaluated. The method and results are shown below.

[0046] First, the skin of an immunodeficient SCID mouse was incised. Then, 16 pieces of the three-dimensional bone-like tissue according to the present embodiment were subcutaneously transplanted into the SCID mouse without using an artificial scaffold material. When the presence or absence of subcutaneous hard tissue was confirmed by micro-CT, as shown in FIG. 8(a), a three-dimensional bone-like tissue exhibiting X-ray opacity was observed 4 weeks after transplantation (white circle in the figure), and as shown in FIG. 9(a), the opacity was improved 8 weeks after transplantation (white circle in the figure).

[0047] Next, the transplanted three-dimensional bone-like tissue was removed together with the skin, decalcified, and then subjected to HE staining and AZAN staining. In AZAN staining, immature bones are stained blue, and mature bones are stained red. As shown in Figure 8(b), bone-like tissue was observed by HE staining 4 weeks after transplantation, but as shown in Figure 8(c), it was shown to be immature bone matrix that stained blue with AZAN staining. On the other hand, 8 weeks after transplantation, tissue that stained red with AZAN staining was observed (Figure 9(c)), along with dark staining with HE staining (Figure 9(b)), confirming that it had become mature bone-like tissue.

[0048] The above results demonstrate that the three-dimensional bone-like tissue according to this example has the ability to autonomously form bone.

[0049] [Example 3: Transplantation of three-dimensional bone-like tissue into calvarial bone defects in immunodeficient mice and rats] Next, to examine the bone regeneration effect of transplanting the three-dimensional bone-like tissue obtained as described above into the bone defect, the effect was evaluated using immunodeficient SCID mice and nude rat calvarial defect models. The method and results are described below.

[0050] (method) First, a bone defect with a diameter of 1.6 mm was created in the calvaria of a SCID mouse using a round burr. Meanwhile, a bone defect with a diameter of 8 mm was created in the calvaria of a nude rat using a trephine burr. Then, three-dimensional bone-like tissue as particles was directly transplanted into the bone defect of the SCID mouse (one piece) and into the bone defect of the nude rat (64 pieces) without using an artificial scaffold material (see FIG. 10). In addition, a case in which three-dimensional bone-like tissue was not transplanted into the bone defect of the SCID mouse was used as a comparative example. Four weeks after transplantation in the SCID mouse and eight weeks after transplantation in the nude rat, the animals were sacrificed, the calvaria was removed, and serial sections were prepared after decalcification in the same manner as above, and HE staining was performed to observe the bone tissue. In addition, the nude rat sample eight weeks after transplantation was also observed by AZAN staining.

[0051] (result) In the SCID mouse model, as shown in FIG. 11, no bone regeneration occurred in the comparative example in which three-dimensional bone-like tissue was not transplanted, whereas bone regeneration was observed in the example in which three-dimensional bone-like tissue was transplanted, as shown in FIG. 12. In addition, in the nude rat model, as shown in FIG. 13, it was observed by HE staining that the defect was filled with bone-like tissue connecting both ends of the defect after transplantation of three-dimensional bone-like tissue. Furthermore, as shown in FIG. 14, most of the defect was stained red by AZAN staining, which confirmed that these bone-like tissues were composed of mature bone. Therefore, it was confirmed that bone regeneration occurred by transplantation of three-dimensional bone-like tissue in the bone defect area.

[0052] From the results of the above Examples 1 to 3, it was revealed that by embedding mesenchymal stem cells in a gel and culturing them in a specific bone induction medium, a cell mass consisting of bone matrix-like tissue containing osteocyte-like cells, i.e., a three-dimensional bone-like tissue, can be obtained without an artificial scaffold material. It was also revealed that transplanting the obtained three-dimensional bone-like tissue into a bone defect site effectively induces bone regeneration.

[0053] As described above, the method for producing bone-like tissue according to the present invention can produce a three-dimensional bone-like tissue consisting of a bone matrix and bone cells that are alive and encapsulated in the bone matrix and form a network with each other. Furthermore, the bone-like tissue according to the present invention obtained by such a method exhibits excellent bone regeneration effects and does not contain an artificial scaffold material, i.e., does not contain a foreign material, and therefore can be suitably used as a transplant material for bone regeneration therapy.

Claims

1. A bone matrix having minerals deposited thereon and bone cells contained within the bone matrix, and no artificial scaffold material is included; The bone cells form a network in an artificial bone-like tissue, The bone-like tissue is a roughly spherical granular tissue that is suspended and cultured in a gel-embedded state, The bone cells are bone cells induced to differentiate from mesenchymal stem cells, The bone matrix is ​​calcium phosphate and contains osteocalcin, osteopontin, osteonectin and collagen. The artificial scaffold material is hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen gel or hyaluronic acid gel,

2. The bone-like tissue of claim 1, which does not contain blood vessels.

3. A pharmaceutical composition for bone regeneration comprising the bone-like tissue according to claim 1 or 2.

4. A method for producing bone-like tissue according to claim 1 or 2, comprising: A step of culturing the three-dimensional mesenchymal stem cell cluster embedded in a gel using an osteogenic differentiation-inducing medium, A method for producing bone-like tissue, characterized in that no artificial scaffold material is used, The bone differentiation induction medium is prepared by adding dexamethasone at a concentration lower than 100 nM, β-glycerophosphate at a concentration lower than 10 mM, and ascorbic acid to high glucose DMEM containing FBS, penicillin, and streptomycin; the artificial scaffold material is hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen gel or hyaluronic acid gel; The method, wherein the gel is a polysaccharide-based gel.

5. A step of obtaining a cell sheet by adhesion culture of mesenchymal stem cells in a culture vessel; detaching the cell sheet from the culture vessel; a step of floating-culture the detached cell sheet to obtain a three-dimensional mesenchymal stem cell cluster; The method for producing bone-like tissue according to claim 4, further comprising:

6. The method for producing bone-like tissue according to claim 4 or 5, characterized in that the gel does not contain any extracellular matrix components other than those generated from the embedded cells.

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