JP7655549B2 - Bone-like tissue and method for producing same - Google Patents

Description

The present invention relates to bone-like tissue and a method for producing the same, in particular to bone-like tissue that does not contain artificial materials and a method for producing the same.

Traditionally, autologous bone grafting, in which bone is isolated from the chin or ilium and transplanted, has been used as a bone regeneration therapy for patients with severe fractures or extensive bone defects following osteotomy due to 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 becomes difficult due to a lack of bone tissue.

On the other hand, mesenchymal stem cells (MSCs), which have multipotency and self-proliferation capabilities, are considered to be suitable for tissue regeneration therapy, and in recent years, many attempts have been made to create and supply three-dimensional bone-like tissue equivalent to autologous bone by mixing MSCs with artificial scaffolding 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).

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, no matter how much MSCs are cultured in a bone differentiation medium, it is impossible to induce them to become bone cells that are contained within the bone matrix and form a network. 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.

To date, representative 3D culture methods for MSCs that do not use artificial materials have attempted to create three-dimensional bone-like tissue by culturing MSC spheroids, which are primarily based on cell-cell adhesion, and three-dimensional cell masses (C-MSCs) constructed from the extracellular matrix (ECM) produced by MSCs, in bone differentiation medium (dexamethasone (100 nM) + β-glycerophosphate (10 mM) + ascorbic acid (50 μg/ml)). However, although mineral deposition could be induced in both MSC spheroids and C-MSCs, appropriate bone matrix formation and induction of bone cells could not be achieved (see non-patent documents 2 and 3).

Amini AR et al., Crit Rev Biomed Eng, 2012, 40(5), 363-408. Langenbach F, Clin Oral Invest, 2013, 17(1), 9-17. Kittaka M et al., Cytotherapy, 2015, 17(7), 860-873.

As mentioned above, the combined use 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.

The present invention has been made in consideration of the above-mentioned problems, and its purpose is to enable MSCs to be cultured three-dimensionally and undergo bone tissue formation without using artificial scaffold materials, thereby obtaining three-dimensional bone-like tissue that is effective for bone regeneration therapy.

In order to achieve the above-mentioned objective, the inventors conducted extensive research and discovered that by embedding three-dimensional mesenchymal stem cell clusters in a gel and culturing them in a bone differentiation-inducing medium, it is possible to produce 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.

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.

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 bone defects, which contains a bone matrix on which minerals are deposited and bone cells contained within the bone matrix, and does not contain foreign matter such as artificial scaffolding materials.

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 suspension-culturing the detached cell sheet to obtain a three-dimensional mesenchymal stem cell cluster.

In the method for producing bone-like tissue according to the present invention, it is preferable that the gel is a gel whose main component is polysaccharide.

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

In addition, in the method for producing bone-like tissue according to the present invention, it is preferable that the bone differentiation induction medium contains ascorbic acid and β-glycerophosphate.

In addition, in the method for producing bone-like tissue according to the present invention, it is preferable that the bone differentiation induction medium contains dexamethasone.

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 within the bone matrix, does not contain an artificial scaffold material, and the bone cells form a network. It is also preferable that the bone-like tissue according to the present invention does not contain blood vessels.

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

The pharmaceutical composition of the present invention is characterized in that it is a pharmaceutical composition for bone regeneration containing the bone-like tissue of the present invention.

The pharmaceutical composition of the present invention contains bone-like tissue as described above, making it suitable for transplantation into bone defects and capable of being effectively used in bone regeneration therapy.

The bone-like tissue and the method for producing the same according to the present invention make it 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.

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 a gel and culturing them in a concentration-optimized bone differentiation-inducing medium. 1 shows photographs showing 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 bone differentiation-inducing medium. Photographs showing 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 concentration-optimized osteodifferentiation-inducing medium. 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 a gel and culturing them in a conventional bone differentiation-inducing medium. This is a photograph obtained by micro-CT photography of a three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing them in a concentration-optimized bone differentiation-inducing medium. (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. 1 shows photographs depicting 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 a concentration-optimized bone differentiation-inducing medium. (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 of 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 AZAN staining of the same section. (a) to (c) are photographs showing the condition of the transplant site 8 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 of 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 AZAN staining of the same section. 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. 1 is an 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. 1 is an HE stained photograph showing the result of transplanting a three-dimensional bone-like tissue into a calvarial bone defect of a SCID mouse 4 weeks after the transplantation. FIG. 1A is an HE-stained photograph of a calvarial bone defect in a nude rat, showing the results 8 weeks after transplanting three-dimensional bone-like tissue into the defect, and FIG. 1B and FIG. 1C are enlarged views of the areas enclosed in the squares in FIG. 1A. FIG. 1A 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 transplantation, and FIG. 1B and FIG. 1C are enlarged views of the areas enclosed in the squares in FIG. 1A.

Hereinafter, the mode for carrying out the present invention will be described with reference to the drawings. The following description of the preferred embodiment is merely exemplary in nature and is not intended to limit the present invention, its application method, or its uses.

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 within 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.

In the bone-like tissue according to one embodiment of the present invention, the bone matrix is an accumulation of substances produced by cells with bone formation ability and secreted outside the cells, including, but 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 bones, and are encapsulated in the bone matrix to form a network with each other and constitute bone together with the bone matrix. Here, network formation 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 protrusions to connect with each other to form a three-dimensional network.

In this embodiment, minerals specifically include calcium, magnesium, and phosphorus. Mineral deposition in this embodiment means a state in which at least calcium is deposited in the bone matrix, and in this case, magnesium or phosphorus may also be deposited in addition. To confirm the presence or absence of mineral deposition, for example, a method of staining tissue sections using alizarin red, a dye that binds to calcium, can be used, but of course this 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.

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

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

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.

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 harvested 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).

In the method for producing bone-like tissue according to the present embodiment, the three-dimensional mesenchymal stem cell cluster is a cell cluster composed of multiple mesenchymal stem cells, and is not a two-dimensional flat sheet-like cell cluster, but a three-dimensional granular cell cluster. Normally, when mesenchymal stem cells are cultured by adhesion, they grow two-dimensionally, that is, in a sheet-like shape to form a cell sheet. However, 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 cluster, and then culture the cell cluster embedded in a gel using a bone differentiation induction 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 specific antibiotic in addition to the above factors can be used. In addition, for example, Prime-XV MSC expansion XSFM (Irvine) which does not contain xenogeneic animal proteins can be used.

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 Bioscience) 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 (extracellular matrix) such as collagen and laminin can also be used, but because they contain a large amount of cell adhesion factors, cells migrate from the cell clusters, and the resulting three-dimensional bone-like tissue becomes small, 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.

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

The pharmaceutical composition of the present invention is a pharmaceutical composition used for bone regeneration including the bone-like tissue of the present invention.

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 limitations. It is preferably applicable to bone regeneration therapy for intractable fractures, periodontitis, or cases of extensive bone loss following osteotomy, although not limited thereto.

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

Below, examples are provided to explain in detail the bone-like tissue and method for producing the same according to the present invention.

[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 on a 96-well plate (manufactured by Corning) at a cell density of 5.0 x 104 cells/well, and cultured for 4 days in a culture medium (GM medium) consisting of High glucose DMEM (manufactured by Sigma) containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, to which 50 μg/ml ascorbic acid (manufactured by Sigma) was added, to produce sufficient ECM. Thereafter, the cell population (cell sheet) that had been attached to the 96-well plate and formed a 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 obtained 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 a diameter of about 600 μm to 800 μm, were obtained.

(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 was added in a 48-well plate, and the cells were 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, while 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. Histological evaluation of the three-dimensional bone-like tissue or cell aggregate obtained in each Example and Comparative Example is described below.

(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 for 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.

As shown in Figure 1, in the Example in which C-MSCs were embedded in gel and cultured in a concentration-optimized bone differentiation induction medium, abundant bone matrix and minerals stained with alizarin red were observed, and in the HE staining image, bone-like matrix stained darkly 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 Figure 2, deposition of minerals stained with alizarin red was observed, but when decalcified sections were 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 the excessive calcification effect of the added factors used for bone differentiation induction. On the other hand, in Comparative Example 2 in which an 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 in the ECM was obtained.

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 in bone differentiation induction was reduced, but in Comparative Examples 1 and 2 in which suspension culture was performed without gel-embedded culture, three-dimensional bone-like tissue was not obtained. 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.

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

As shown in FIG. 5, the three-dimensional bone-like tissue of this example showed X-ray opacity overall, similar to human bone tissue. Furthermore, in the three-dimensional bone-like tissue of this example, as shown in FIGS. 6(a) to (c), expression of COL1, OPN, and OCN was observed in the area stained darkly with HE shown in FIG. 1 (the white area inside the three-dimensional bone-like tissue in FIGS. 6(a) to (c)). Furthermore, as shown in FIG. 7, in the three-dimensional bone-like tissue of this example, 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 believed that a three-dimensional bone-like tissue with characteristics equivalent to those of human bone tissue was obtained in this example.

[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.

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 using micro-CT, as shown in FIG. 8(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 had improved 8 weeks after transplantation (white circle in the figure).

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 addition, immature bones are stained blue with AZAN staining, and mature bones are stained red. As shown in Figure 8(b), bone-like tissue was observed with HE staining four 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, eight 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.

The above results demonstrate that the three-dimensional bone-like tissue of this embodiment has the ability to form bone autonomously.

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

(method)
First, a bone defect with a diameter of 1.6 mm was created in the calvaria of a SCID mouse using a round bar. Meanwhile, a bone defect with a diameter of 8 mm was created in the calvaria of a nude rat using a trephine bar. 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. After 4 weeks of transplantation in the SCID mouse and 8 weeks of 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 after 8 weeks of transplantation was also stained with AZAN and observed.

(result)
In the SCID mouse model, bone regeneration did not occur in the comparative example in which three-dimensional bone-like tissue was not transplanted as shown in Figure 11, whereas bone regeneration was observed in the example in which three-dimensional bone-like tissue was transplanted as shown in Figure 12. In the nude rat model, as shown in Figure 13, it was observed that the defect was filled with bone-like tissue connecting both ends by transplantation of three-dimensional bone-like tissue, as shown in HE staining. As shown in Figure 14, most of the area 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 occurs by transplantation of three-dimensional bone-like tissue in the bone defect area.

The results of Examples 1 to 3 above demonstrated 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., three-dimensional bone-like tissue, can be obtained even without an artificial scaffold material. It was also demonstrated that transplanting the obtained three-dimensional bone-like tissue into a bone defect effectively induces bone regeneration.

As described above, the method for producing bone-like tissue according to the present invention makes it possible to obtain three-dimensional bone-like tissue consisting of bone matrix and bone cells that are surviving 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 artificial scaffold materials, i.e., does not contain foreign materials, and therefore can be suitably used as a transplant material for bone regeneration therapy.

Claims (6)

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,
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,
An artificial bone-like tissue, wherein the bone matrix is calcium phosphate and contains osteocalcin, osteopontin, osteonectin and collagen, and the artificial scaffold material is hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen gel or hyaluronic acid gel. The bone-like tissue according to claim 1, characterized in that it does not contain blood vessels. A pharmaceutical composition for bone regeneration comprising the bone-like tissue according to claim 1 or 2. A method for producing bone-like tissue according to claim 1 or 2, comprising:
Cultivating 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. 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;
The method for producing bone-like tissue according to claim 4, further comprising 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 or 5, characterized in that the gel does not contain extracellular matrix components other than those generated by the embedded cells.

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