JP6901727B2 - Complex of undifferentiated osteoblasts derived from alveolar bone and carrier for undifferentiated osteoblasts derived from alveolar bone and utilization thereof - Google Patents

Description

The present invention relates to a complex of undifferentiated osteoblasts derived from alveolar bone and a carrier for undifferentiated osteoblasts derived from alveolar bone, and its utilization.

Periodontal disease is an infectious disease that causes tooth loss due to inflammatory collapse of periodontal tissue, and the number of patients in Japan is said to be 50 million. Periodontal disease is said to be the biggest cause of deterioration of oral function, and periodontal disease bacteria and inflammatory substances that cause periodontal disease cause diseases such as cardiovascular disease, diabetes, and respiratory disease. It has also been reported to bring about progress. From this, treatment for periodontal disease and recovery from periodontal disease, which have been clarified to be one of the causes that impair not only the oral health but also the health of the whole body, are regarded as important.

In addition, periodontal disease is an infectious disease that should be noted regardless of age, and middle-aged and older people in their 40s and older, especially those in their 50s and older, are a high-risk group of severely advanced invasive periodontal disease. It has been known. As a countermeasure against such serious periodontal disease, the development of regenerative medicine technology for restoring periodontal tissue is required.

As a conventional periodontal tissue regenerative medicine technique, artificial fixation to a damaged part of bone tissue, transplantation of a bone filling agent, or autologous bone has been adopted. However, these methods have a problem that the bone tissue cannot be sufficiently restored to a normal state and it takes a long period of time. In order to overcome these problems, cell transplantation technology using mesenchymal stem cells having bone regeneration ability has been developed. However, mesenchymal stem cells cannot be said to be a useful technique because there are individual differences in bone regeneration ability and it is difficult to obtain them from middle-aged and elderly people. In addition, methods for transplanting bovine bone incineration products at about 300 ° C., human decalcified freeze-dried bone, etc. are also known, but there are concerns about their use due to the risk of prion-derived mad cow disease and unknown virus infection. ing.

Further, in regenerative medicine, a method is known in which cells are supported on a carrier such as a polymer gel or a ceramic material, and the obtained carrier is transplanted. However, there are problems that cells do not proliferate sufficiently on the carrier, do not differentiate, and have poor adaptability to the complex morphology of the transplant destination, and clinically practically useful techniques have been constructed. It's hard to say.

On the other hand, the present inventors have succeeded in obtaining undifferentiated osteoblasts derived from alveolar bone (Patent Document 1).

International Publication No. 2010/021162

Therefore, the present invention uses a composite of undifferentiated osteoblasts derived from alveolar bone and a carrier useful for bone tissue regeneration using the undifferentiated osteoblasts derived from alveolar bone, and bone tissue using the composite. The main purpose is to provide a method for reproduction and the like.

As a result of diligent studies in view of the above problems, the present inventors have found that undifferentiated osteoblasts derived from alveolar bone can be efficiently proliferated and / or differentiated by using a specific carrier described later. In addition, the present inventors have found that by using the carrier in combination with undifferentiated osteoblasts derived from alveolar bone, bone tissue can be regenerated to a clinically practical level. The present invention has been completed as a result of further studies based on the above findings, and is as follows.
Item 1. Complex of undifferentiated osteoblasts derived from alveolar bone and carrier:
Here, the carrier is a fiber molded product having a three-dimensional network structure, the bulk density of the fiber molded product is 0.0001 to 0.25 g / cm ³ , and the fibers constituting the fiber molded product are biocompatible. Contains sex polymers.
Item 2. Item 2. The composite according to Item 1, wherein the fibers constituting the fiber molded product contain a biocompatible hydrophobic polymer.
Item 3. Item 2. The composite according to any one of Items 1 or 2, wherein the fibers constituting the fiber molded product include composite fibers containing a biocompatible hydrophobic polymer and a biocompatible hydrophilic polymer.
Item 4. Item 3. The composite according to Item 3, wherein the composite fiber has a structure in which the hydrophilic polymer fiber is formed inside the hydrophobic polymer fiber.
Item 5. The fibers of the hydrophilic polymer form a three-dimensional network structure inside the composite fiber, and the fibers of the hydrophilic polymer are bonded to each other to form a twisted structure in the composite fiber to bring the composite fibers into contact with each other. Item 4. The composite according to Item 4, which is controlled.
Item 6. Item 2. The composite according to Item 1 to 5, wherein the average diameter of the fibers constituting the fiber molded product is 0.05 to 30 μm.
Item 7. Item 2. The composite according to any one of Items 1 to 6, wherein the fiber molded product has a coating layer at least in part.
Item 8. Item 2. The composite according to Item 7, wherein the coating layer has a thickness of 1000 μm or less.
Item 9. Item 7. The composite according to Item 7 or 8, wherein the coating layer has a plurality of through holes having an average pore diameter of 10 μm or more.
Item 10. Item 9. The composite according to Item 9, wherein the area of the opening formed by the plurality of through holes is 50% or more of the surface area of the coating layer.
Item 11. Item 2. The composite according to any one of Items 1 to 10, wherein the thickness of the carrier is 0.05 to 500 mm.
Item 12. Item 1 in which the fiber constituting the fiber molded product contains at least one hydrophobic polymer selected from the group consisting of polylactic acid, polycaprolactone, polyglycolic acid and a copolymer composed of two or more of these. The composite according to any one of 11 to 11.
Item 13. Differentiation of undifferentiated osteoblasts derived from alveolar bone into osteoblasts as compared with the case where the carrier 2 (bulk density 0.21 g / cm ^{3, fiber-forming body having a thickness of about 80 μm) shown in Examples is used.} The complex according to any one of Items 1 to 12, wherein the differentiation of undifferentiated osteoblasts into osteoblasts is evaluated using alkaline phosphatase staining as an index, and alkaline phosphatase staining is used as an index. Incubates the complex in an MF medium (manufactured by Toyo Boseki Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone in the presence of 5% CO2 at 37 ° C. and in 10 wt% formaldehyde. It is fixed for 30 minutes and stained with an alkaline phosphatase stain for 5 minutes.
Item 14. Items 1 to 1, wherein the carrier can promote calcification in the composite as compared with the case where the carrier 2 (bulk density 0.21 g / cm ^{3, fiber-forming body having a thickness of about 80 μm) shown in Examples is used.} The complex according to any of 13; here, calcification is evaluated using alizarin red staining as an index, and alizarin red staining is performed by converting the complex to 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, 100 nmol /. l Incubate in MF medium containing dexamethasone (manufactured by Toyo Boseki Co., Ltd.) in the _{presence of 5% CO 2} at 37 ° C., fix with 10 wt% formaldehyde for 30 minutes, and stain with alizarin red for 5 minutes.
Item 15. Item 2. The complex according to any one of Items 1 to 14, wherein at least a part of the undifferentiated osteoblasts derived from alveolar bone is in a state of being differentiated into osteoblasts derived from alveolar bone.
Item 16. Item 2. The complex according to any one of Items 1 to 14, wherein at least a part of the undifferentiated osteoblasts derived from the alveolar bone is in a calcified state.

According to the present invention, undifferentiated osteoblasts derived from alveolar bone can be efficiently proliferated and / or differentiated on a carrier. Further, according to the present invention, bone tissue derived from undifferentiated osteoblasts derived from alveolar bone can be easily and efficiently produced. According to the present invention, bone tissue can be regenerated to a practical extent.

FIG. 1 is an example of a composite fiber containing a hydrophobic polymer and a hydrophilic polymer in which a twisted structure is formed. FIG. 2 is an example in which only the hydrophobic polymer is etched with a solvent among the composite fibers containing the hydrophobic polymer and the hydrophilic polymer, and the structure of the hydrophilic polymer in the composite fiber is visualized. FIG. 3 is an example of a composite fiber containing a hydrophobic polymer and a hydrophilic polymer in which a twisted structure is formed. FIG. 4 is a model diagram in which the carrier of the present invention includes a coating layer. It is a model diagram of a fiber molded body in which a coating layer (shape stabilizing layer) is formed on the surface of a cotton-like fiber molded body. FIG. 5 is an enlarged photograph of the surface of the coating layer. FIG. 6 is a model diagram of a fiber molded body in which a coating layer and a high-density layer are formed on the surface of the cotton-like fiber molded body. FIG. 7 is a model diagram of a fiber molded product in which a high-density layer is formed inside the cotton-like fiber molded product and a coating layer is formed on the surface thereof. FIG. 8 is a model diagram of a fiber molded body in which a high-density layer is formed inside and on the surface of the cotton-like fiber molded body and a coating layer is formed on the surface. FIG. 9 is a model diagram of a cotton-like fiber molded product containing a different optional component, having a different polymer composition, or having both characteristics, and having a coating layer formed on the surface. FIG. 10 shows a cotton-like fiber molded product containing different optional components, or having different polymer compositions, or having both of these characteristics, containing different optional components, or having different polymer compositions. Alternatively, it is a model diagram of a fiber molded body in which a high-density layer having both of these characteristics is formed and a coating layer is formed on the surface. FIG. 11 is a model diagram of a fiber molded product in which a plurality of carriers having different compositions or internal structures are bonded. FIG. 12A) shows the results of measuring the PD of HAOB obtained from the alveolar bone of each age group. In addition, B) of FIG. 12 shows the results of karyotyping of HAOB3 (PD35) induced in interphase by the G-band method (top) and the SKY method (bottom). FIG. 13 shows the results of culturing HAOB3 in a medium supplemented with various growth factors and evaluating the cell proliferation activity. In the figure, the vertical axis shows the relative value of cell proliferation activity calculated with the number of cells when cultured using DMEM without serum added as 1. In the figure, "MF" is MF medium, "20% FCS" is DMEM medium containing 20% by volume FCS, "PDGFAA" is DMDM medium containing 10 ng / ml PDGFAA, and "PDGFAB" is 10 ng / ml. PDGFAB is DMEM medium supplemented with PDGFAB, “PDGFBB” is DMEM medium supplemented with 10 ng / ml PDGFBB, and “bFGF” is DMEM medium supplemented with 10 ng / ml bFGF. A) of FIG. 14-1 shows the results of alizarin red staining (left) and the results of measuring alkaline phosphatase activity (right) after culturing HAOB3 in hBMP-2 added medium or MF medium. In addition, B) in FIG. 14-1 shows the results of measuring the intracellular expression levels of RUNX2, OSTERIX, OCN and BSP after culturing HAOB3 in hBMP-2 added medium or MF medium. The vertical axis in B) shows the relative value of the expression level of each differentiation marker calculated by setting the expression level of β-Actin to 100. FIG. 14-2 shows the results of culturing HAOB3 in hBMP-2 supplemented medium or MF medium and then staining with an antibody against OPN and an antibody against OCN. A) of FIG. 15 shows the results of alizarin red staining after culturing HAOB3 of 6PD to 29PD. FIG. 15B) shows the results of measuring the intracellular OCN expression level after culturing HAOB3 such as 6PD in a medium containing hBMP-2. FIG. 15C) shows the results of measuring the intracellular expression levels of RUNX2, OSTERIX and BSP after culturing HAOB3 such as 6PD in a medium containing hBMP-2. The vertical axis in B) and C) shows the relative value of the expression level of each differentiation marker calculated by setting the expression level of β-Actin to 100. In A) of FIG. 16, after culturing HAOB3 and NHOst in a medium containing hBMP-2 at a concentration of 50 to 1000 ng / ml, the result of alizarin red staining (left) and the result of measuring alkaline phosphatase activity (right). Is shown. FIG. 16B) shows the results of measuring the intracellular expression levels of RUNX2, OSTERIX, OCN and BSP after culturing HAOB3 and NHOst in a medium containing hBMP-2 at a concentration of 100 ng / ml. The vertical axis in B) shows the relative value of the expression level of each differentiation marker calculated by setting the expression level of β-Actin to 100. In FIG. 17, a shows the OPN of the differentiated HAOB3, b shows the OCN of the differentiated HAOB3, c shows the superimposed image of a and b, and d shows the differentiated NHOst. As a result of staining the OPN of, e is the result of staining the OCN of the differentiation-induced NHOst, and f is an image in which d and e are superimposed. FIG. 18 shows the results of measuring the expression level of BMP-2 after culturing HAOB3 and NHOst in a medium containing hBMP-2 at a concentration of 100 ng / ml. The vertical axis shows the relative value of the expression level of hBMP- calculated by setting the expression level of β-Actin to 100. A) of FIG. 19 shows the results of measuring the expression levels of MGP, STMN2, and NEBL in HAOB3 (6PD and 35PD). The vertical axis in A) shows the relative value of the expression level of each gene calculated by setting the expression level of β-Actin to 100. B) shows the results of measuring the expression levels of RUNX2, OSTERIX, OCN, BSP, MGP, STMN2, and NEBL in HAOB3 (6PD). The vertical axis in B) of FIG. 19 shows the relative value of the expression level of each gene calculated by setting the expression level of β-Actin to 100. FIG. 19C) shows the gene expression change rates (%) of RUNX2, OSTERIX, OCN, BSP, MGP, STMN2, and NEBL in 6PD and 35PD of HAOB3. This gene expression change rate is calculated by multiplying the value obtained by dividing the gene expression level of HAOB3 in 6PD by the gene expression level of HAOB3 in 35PD by 100. FIG. 20 shows the results of measuring the expression levels of STMN2, NEBL and MGP in HAOB1-4, human epidermis-derived fibroblasts (HFF), human osteosarcoma cells (MG63), and femur-derived osteoblasts (NHOst). Shown. The vertical axis in each figure shows the relative value of the expression level of each gene calculated by setting the expression level of β-Actin to 100. FIG. 21 shows the proliferation of alveolar bone-derived undifferentiated osteoblasts on a carrier. FIG. 22 shows the results showing changes in alkaline phosphatase activity. FIG. 23 shows the time course of calcium deposition by alizarin red staining. FIG. 24 shows the results showing changes in the expression of calcification-related genes in the complex. FIG. 25 shows the proliferation of alveolar bone-derived undifferentiated osteoblasts on a carrier. FIG. 26 shows changes in alkaline phosphatase activity and changes in calcium deposition due to alizarin red staining. FIG. 27 shows the formation of bone-like structures in vivo.

Hereinafter, the present invention will be described.
Complex of undifferentiated osteoblasts derived from alveolar bone and carrier The present invention relates to a complex of undifferentiated osteoblasts derived from alveolar bone and a carrier.

Undifferentiated osteoblasts derived from alveolar bone In the present invention, undifferentiated osteoblasts derived from alveolar bone are described as follows.

In the present invention, the undifferentiated osteoblast means a pre-osteoblast that has not differentiated into an osteoblast, has a proliferative ability, and has an ability to differentiate into an osteoblast. Here, "having a proliferative ability" means that undifferentiated osteoblasts, which will be described later, can divide and proliferate in a proliferative medium. Further, "having the ability to differentiate into osteoblasts" has the property of differentiating into osteoblasts in the presence of an osteoblast-inducing factor such as BMP2 or in a state of being supported on a carrier described later. Indicates that When differentiated into osteoblasts, for example, increased alkaline phosphatase activity, increased cell staining intensity by Alizarin red staining, and osteoblast differentiation markers (RUNX2, OSTERIX, OSTEOCALCIN (OCN), BONE SIALOPROTEIN (BSP), Since an increase in the expression level of OSTEOPONTIN (OPN)) is observed, whether or not it has differentiated into osteoblasts is judged using these as indicators.

The alveolar bone can be easily understood by those skilled in the art, for example, can be collected by a usual surgical method, and the alveolar bone removed at the time of tooth extraction can be conveniently used.

The alveolar bone-derived undifferentiated osteoblasts used in the present invention are obtained by subjecting the alveolar bone to enzymatic treatment to obtain alveolar bone-derived cells, and then culturing the undifferentiated osteoblasts in a medium capable of proliferating. Obtainable.

Here, in order to obtain cells derived from alveolar bone by enzymatically treating the alveolar bone, the enzyme may act on the alveolar bone in a buffer solution such as a phosphate buffer solution. As the enzyme used to obtain cells derived from alveolar bone from alveolar bone, an enzyme generally used when separating cells from a living tissue piece may be used. Specifically, collagenase, Examples thereof include proteases such as pepsin and trypsin. Among these enzymes, coragenase is preferable from the viewpoint of efficiently recovering cells from the alveolar bone.

Further, in order to increase the enzyme treatment efficiency, the collected alveolar bone may be cut into 5 to 10 mm pieces prior to the enzyme treatment.

The conditions under which the enzyme acts on the alveolar bone are not particularly limited as long as the cells derived from the alveolar bone can be released, and examples thereof include the following conditions.
Alveolar bone concentration: The concentration of alveolar bone is set so as to be 1 ml for three alveolar bone fragments, preferably 0.5 to 1.5 ml for one alveolar bone fragment.
Enzyme concentration: For example, when collagenase (> 1.5 U / mg) is used, the enzyme concentration is set to 1 to 4 mg / ml, preferably 1.5 to 3 mg / ml. In addition, in this specification, collagenase 1U is 1μ from 4-phenyl-azobenxyl-oxycarbonyl-L-prolyl-L-leucyl-L-glycyl-L-prolyl-D-arginine at 25 ° C. for 1 minute. Represents the enzyme titer capable of liberating a mole of 4-phenyl-azobenxyl-oxycarbonyl-L-prolyl-L-leucine.
Processing temperature: Set to 37 ° C ± 2 ° C.
Treatment time: Set to 10-40 minutes, preferably 15-25 minutes.

The enzyme treatment on the alveolar bone may be repeated a plurality of times as necessary in order to increase the recovery rate of cells derived from the alveolar bone, preferably 4 to 12 times.

By enzymatic treatment in this way, cells derived from alveolar bone are released from the alveolar bone. Therefore, after the enzyme treatment, the cells derived from the alveolar bone can be obtained by performing a known means such as centrifugation.

By culturing the alveolar bone-derived cells thus obtained in a medium in which undifferentiated osteoblasts can proliferate, an alveolar bone-derived undifferentiated osteoblast group can be obtained.

As a medium capable of proliferating undifferentiated osteoblasts, a growth factor required for the proliferation of undifferentiated osteoblasts is used in a basal medium containing inorganic salts, amino acids, vitamins, etc., which are essential components for proliferating animal cells. And those to which an additive component is added. Examples of such basal medium include Eagle's basal medium (MEM), α-Eagle's basal medium (αMEM), Dulbecco's modified Eagle's medium (DMEM), Ham F-12 medium and the like. As long as undifferentiated osteoblasts derived from alveolar bone can be obtained, a suitable example of a medium capable of proliferating the undifferentiated osteoblasts is a medium containing PDGF (platelet-derived growth factor) as a growth factor. .. The presence of PDGF makes it possible to efficiently proliferate undifferentiated osteoblasts having excellent bone tissue regeneration ability from cells derived from alveolar bone. PDGF may be contained in the medium, or may be contained in the medium by external addition.

PDGF can be divided into PDGFAA, PDGFAB, PDGFBB, etc. depending on the combination of subunits, but any of them may be used in the present invention. Further, the origin of PDGF is not particularly limited, and it may be derived from animal tissue, produced by gene recombination technique, or the like. The concentration of PDGF in the medium is exemplified by 1 to 100 ng / ml, preferably 10 to 30 ng / ml.

Further, as a medium in which undifferentiated osteoblasts can proliferate, it is preferable that monoethanolamine is contained in addition to PDGF. When monoethanolamine is contained, the concentration of monoethanolamine in the medium is exemplified by 0.1 to 100 μg / ml, preferably 6 μg / ml.

Further, the medium may contain one or more of insulin, transferrin, and bFGF (basic fibroblast growth factor). When insulin is added to the medium, the concentration of insulin in the medium is exemplified by 1 to 100 μg / ml, preferably 10 μg / ml. When transferrin is added to the medium, the concentration of transferrin in the medium is exemplified by 1 to 100 μg / ml, preferably 5 to 15 μg / ml. When bFGF is added to the medium, the concentration of bFGF in the medium is exemplified by 1 to 100 ng / ml, preferably 5 to 15 ng / ml.

In addition, the medium in which undifferentiated osteoblasts can proliferate may contain antibiotics such as streptomycin, kanamycin, and penicillin.

From the viewpoint of easily proliferating undifferentiated osteoblasts, an MF medium (manufactured by Toyobo Co., Ltd.) is exemplified as a suitable specific example of a medium in which undifferentiated osteoblasts can proliferate.

To obtain undifferentiated osteoblasts derived from alveolar bone, cells derived from alveolar bone are added to a medium in which undifferentiated osteoblasts can proliferate, and 1 to 7 _{are obtained, for example, at 37 ° C. and 5% CO 2 conditions.} It may be cultured for 1 day, preferably 4 days.

By culturing in this way, the proliferated undifferentiated osteoblasts exist in a state of being attached to the base of the culture vessel. Therefore, by collecting the cells attached to the base of the culture vessel, the alveolar used in the present invention. Undifferentiated osteoblasts derived from bone can be obtained. In particular, since the proliferated undifferentiated osteoblasts exist in a state of being attached to the base of the culture vessel in this way, the alveolar bone derived from the alveolar bone used in the present invention is obtained by collecting the cell group attached to the base of the culture vessel. Undifferentiated osteoblasts can be efficiently obtained.

As a method for recovering the cells attached to the base of the culture vessel, a known method may be followed. For example, 0.25% trypsin and 1 mM EDTA (ethylenediaminetetraacetic acid) may be allowed to act on the cells attached to the base of the culture vessel. Just do it. From this, according to the present invention, it is possible to obtain a cell group having a bone tissue regenerating ability obtained by culturing undifferentiated osteoblasts collected from alveolar bone.

Further, in the above description, the collected alveolar bone tissue is enzymatically treated to obtain undifferentiated cells derived from alveolar bone in the first step, and the undifferentiated cells derived from alveolar bone obtained in the first step are undifferentiated. The second step of culturing the osteoblasts in a proliferative medium, and the undifferentiated osteoblasts and / or cell groups derived from the alveolar bone (that is, the undifferentiated osteoblasts derived from the alveolar bone (cells)) proliferated in the second step. It can also be said to explain a method for producing undifferentiated osteoblasts (cell group) derived from alveolar bone having bone tissue regeneration ability, which includes a third step of recovering group)).

The alveolar bone-derived undifferentiated osteoblasts used in the present invention are significantly superior in bone tissue regeneration ability as compared with conventionally known osteoblasts. Therefore, by using undifferentiated osteoblasts (cell group) derived from alveolar bone, and particularly by using the undifferentiated osteoblasts (cell group) together with a carrier described later, clinically practical bone tissue regeneration can be achieved. It will be possible.

The undifferentiated osteoblasts are also characterized in that they can be subcultured for a long period of time (for example, up to 30 Population Doubling). In the subculture, the cells may be cultured according to a conventionally known procedure using a medium capable of proliferating the undifferentiated osteoblasts described above. For example, as in the examples described later, the undifferentiated osteoblasts described above proliferate. The medium may be changed every 3 days in a possible medium.

The alveolar bone-derived undifferentiated osteoblasts used in the present invention include MGP (matrix gla protein: NM_000900.3 (National center for biotechnology (NCBI))) and STMN2 (stathmin-like 2: NM_007029.3 (NCBI). )) And NEBL (nebulette: NM_006393.2 (NCBI)) are also expressed. These genes are hardly expressed or expressed in osteoblasts derived from the femur, and the present invention It is highly expressed in alveolar bone-derived undifferentiated osteoblasts used in, and can be used as a novel marker peculiar to alveolar bone-derived undifferentiated osteoblasts. More specifically, β-Actin expression. Relative values with an amount of 100 or more, MGP of 70 or more, NEBL of 50 or more, and STMN2 of 50 or more are identified as alveolar bone-derived undifferentiated osteoblasts used in the present invention. The expression levels of these genes are measured by conventionally known methods such as gene chip analysis, RT-PCR method, and real-time PCR method. As methods for measuring the expression levels of these genes, gene chip analysis and real-time PCR are used. The method is preferable. As the gene chip used for gene chip analysis, a commercially available product (for example, HT Human Genome U133 Array Plate Set (Gene Chip; Affymetrix, CA, USA)) may be used or is known. (Lipshutz, RJ et al., (1999) Nature genet. 21, Suppliment, 20-24) may be used.

Furthermore, the undifferentiated osteoblasts are also characterized in that they are induced to differentiate into osteoblasts in vitro in the presence of BMP-2 (bone morphogenetic protein-2). In this case, in order to differentiate the undifferentiated osteoblasts into osteoblasts, specifically, the undifferentiated osteoblasts are prepared in a medium containing BMP-2, dexamethasone, ascorbic acid, and β-glycerophosphate. Should be cultivated. Furthermore, the undifferentiated osteoblasts also have a feature of secreting BMP-2 by inducing differentiation in the presence of BMP-2. Such a feature is one proof that the undifferentiated osteoblasts derived from alveolar bone are different from the conventionally known osteoblasts. Furthermore, when the undifferentiated osteoblasts are differentiated into osteoblasts, for example, an increase in alkaline phosphatase activity, an increase in cell staining intensity by Alizarin red staining, and osteoblast differentiation markers (RUNX2, OSTERIX, OSTEOCALCIN (RUNX2, OSTERIX, OSTEOCALCIN) Increased expression levels of OCN), BONE SIALOPROTEIN (BSP), OSTEOPONTIN (OPN)) are observed. The expression levels of these can be known according to a conventionally known procedure.

Carrier for undifferentiated osteoblasts derived from alveolar bone In the present invention, the carrier for undifferentiated osteoblasts derived from alveolar bone is described as follows.

In the present invention, the carrier is a fiber molded product having a three-dimensional network structure, the bulk density of the fiber molded product is 0.0001 to 0.25 g / cm ³ , and the fibers constituting the fiber molded product are biocompatible. Contains sex polymers.

In the present invention, the fiber molded product having a three-dimensional network structure means an agglomerate of fibers, and one or a plurality of fibers are laminated, woven, knitted, or formed by another method. Refers to the porous molded body of. Although not limiting the present invention to this extent, examples of the form of the fiber molded product include any form such as a cotton-like fiber molded product, a non-woven fabric-like fiber molded product, and a mixed fiber molded product thereof. .. Further, as the shape of the fiber molded product, although not limiting the present invention, for example, any shape such as a block shape, a columnar shape, a plate shape, a tube shape, a spherical shape, and a fixed bulk shape can be mentioned.

In the present invention, the bulk density of the fibrous molded product is 0.0001 to 0.25 g / cm ³ , preferably 0.001 to 0.1 g / cm ³ , and good retention of the three-dimensional morphology, the undifferentiated bone. from the viewpoint of the efficiency of differentiation proliferation efficiency and / or the undifferentiated osteoblasts blasts, and more preferably 0.001-0.05 grams / cm ^3, more preferably 0.002~0.03g / cm ³ is illustrative Will be done. Here, the bulk density is calculated using the volume and weight of the carrier (fiber molded product) measured at room temperature (25 ° C.). Here, the volume is calculated by cutting out a fiber molded product into a rectangular parallelepiped (about 8 cm ³ ), measuring its outer shape directly using a ruler, caliper, etc., and multiplying it by its length, width, and height, or taking a picture. It is obtained by measuring and calculating the outer shape obtained from the obtained image or the like by image analysis. In the present invention, the bulk density means an average value of three or more points of the bulk density measured as described above.

The fibers constituting the fiber molded product are not limited as long as they contain a biocompatible polymer. In the present invention, the biocompatible polymer means a polymer that has no or little adverse effect on the living body when applied to the living body and can be used in the living body for a long period of time, and is a biodegradable polymer or bioabsorbable. Polymers and the like are exemplified.

Examples of the polymer include lactides and lactones (for example, β-propiolactone, β-butyrolactone, β-pivalolactone, γ-butyrolactone, δ-valerolactone, β-methyl-δ-valerolactone, ε-caprolactone, etc.). , Glycolide-containing glycolic acids, ethylene oxalate (1,4-dioxane-2,3-dione), carbonates (eg trimethylin carbonate, etc.), ethers (eg 1,3-dioxane, etc.), ether esters (eg, 1,3-dioxane, etc.) Cyclic monomers such as dioxanone), lactams (εcaprolactam, etc.); hydroxycarboxylic acids such as lactic acid, glycolic acid, 3-hydroxypropanoic acid, 3-hydroxybutanoic acid, 4-hydroxybutanoic acid, 6-hydroxycaproic acid, etc. Or an alkyl ester thereof; a substantially equimolar mixture of an aliphatic diol such as ethylene glycol or 1,4-butanediol and an aliphatic dicarboxylic acid such as succinic acid or adipic acid or an alkyl ester thereof; Examples thereof include single or copolymers of ester monomers, aliphatic polyester resins such as block copolymers, and the like. These can be referred to as biocompatible hydrophobic polymers. As the polymer, preferably, polylactic acid, polycaprolactone, polyglycolic acid, a copolymer composed of two or more of them, and the like are exemplified, and more preferably, polyL-lactic acid, polyDL-lactic acid, polyD-lactic acid, and the like. Examples thereof include poly-p-dioxanone, poly L-lactic acid / ε-caprolactone copolymer, poly L-lactic acid / glycolic acid copolymer, poly DL-lactic acid / glycolic acid copolymer and the like. These may be used alone or in combination of two or more.

Further, the fibers constituting the fiber molded product may further contain a biocompatible hydrophilic polymer. Examples of the hydrophilic polymer include polysaccharides such as cellulose, agarose, alginic acid and gums, derivatives of polysaccharides such as methyl cellulose, propyl cellulose and benzyl cellulose, acetate fibers such as cellulose diacetate and cellulose triacetate, heparin or its derivatives, and chondroitin. Alternatively, its derivatives, glycosaminoglycans such as hyaluronic acid, mucopolysaccharides such as chitin and chitosans or derivatives thereof, collagens such as atelopeptide collagen and reconstituted fiber collagen or derivatives thereof, gelatin, fibroin, keratin, polyglutamic acid or Examples thereof include polypeptides such as the salts thereof, or mixtures, cross-links, and complexes composed of two or more kinds of these polymers. Regarding naturally-derived polymers, properties such as molecular weight distribution and isoionic points differ depending on the production method, but if the polymer solution can be prepared to have a predetermined viscosity in obtaining the fiber molded product, it is suitable for the purpose and the like. May be used as appropriate. As the polymer, gelatin, collagen, polyglutamic acid and a salt thereof, and agarose are preferably exemplified, and more preferably gelatin, collagen, polyglutamic acid, and sodium polyglutamic acid are exemplified. These may be used alone or in combination of two or more.

When the fibers constituting the fiber molded product contain a hydrophilic polymer, the hydrophilic polymer and the hydrophobic polymer may form fibers in which the hydrophilic polymer and the hydrophobic polymer are randomly or uniformly mixed, and the hydrophilic polymer and the hydrophobic polymer may be formed. The polymer may form the fibers substantially without mixing, or the hydrophilic polymer may be present on at least a portion of the surface of the hydrophobic polymer to form the fibers. The fibers that make up the fiber molding may be any mixture of these fibers. For example, the fiber molded product is both a fiber formed by randomly or uniformly mixing a hydrophilic polymer and a hydrophobic polymer, and a fiber formed by substantially mixing the hydrophilic polymer and the hydrophobic polymer. The fiber molded product may include one or both of the above-mentioned fibers, and a hydrophilic polymer may be further present in at least a part thereof.

Although not limiting the present invention, in the present invention, in order to regenerate the bone tissue of the affected area, undifferentiated osteoblasts derived from alveolar bone (at least a part of them may be differentiated into osteoblasts and are calcified. It is desirable that the artificial bone material containing the composite of the carrier and the carrier has a three-dimensional shape, and from this viewpoint, the carrier is three-dimensionally formed, and the carrier is formed even during cell culture and differentiation. It is more preferable that the three-dimensional shape can be stably maintained. From the viewpoint of maintaining the three-dimensional shape of the carrier more stably, the fibers of the fiber molded body constituting the carrier form composite fibers in which the fibers of the hydrophilic polymer are formed inside the fibers of the hydrophobic polymer. Is more preferable, and the fibers of the hydrophilic polymer form a three-dimensional network structure inside the composite fiber, and the fibers of the hydrophilic polymer are bonded to each other to form a twisted structure in the composite fiber to bring the composite fibers into contact with each other. It is more preferred to control and stabilize the three-dimensional shape of the carrier. In this case, the fiber diameter of the hydrophilic polymer fiber is preferably 10 to 1000 nm in order to form a twisted structure in the composite fiber.

Although not limiting the present invention, such fibers are illustrated in FIGS. FIG. 1 is a composite fiber containing a hydrophobic polymer and a hydrophilic polymer having a twisted structure. The twisting of the fibers limits the contact between the composite fibers and contributes to stabilizing the three-dimensional shape of the carrier. FIG. 2 shows a visualization of the structure of the hydrophilic polymer in the composite fiber by etching only the hydrophobic polymer among the composite fibers containing the hydrophobic polymer and the hydrophilic polymer with a solvent. The hydrophilic polymer fibers form a three-dimensional network structure inside the composite fibers, and the hydrophilic polymer fibers are bonded to each other. FIG. 3 is an example of a model diagram of such a composite fiber. Although not limiting the present invention, FIG. 3 is an example in which a part of the surface of the composite fiber is coated with a hydrophilic polymer.

Although not limited as long as the effects of the present invention can be obtained, the content of the biocompatible polymer (total amount of the hydrophobic polymer and the hydrophilic polymer) in the fibers constituting the fiber molded product is preferably 50% by weight or more, more preferably 50% by weight or more. Is exemplified by 70 to 100% by weight, more preferably 90 to 100% by weight, and particularly preferably substantially 100% by weight.

Further, although not limited as long as the effects of the present invention can be obtained, among the fibers constituting the fiber molded product, the composite fiber containing the hydrophobic polymer of the biocompatible polymer and the hydrophilic polymer is preferably 50% by weight of the hydrophobic polymer. As described above, it is exemplified that the hydrophobic polymer is more preferably 70% by weight or more, and particularly preferably the hydrophobic polymer is 80 to 99.5% by weight.

The fiber may contain any component such as a polymer other than the biocompatible polymer, if necessary, as long as the effects of the present invention can be obtained. Examples of the component include surfactants, chemicals, and fillers (polymer particles, metal particles, ceramic particles, etc.).

Of any component, the surfactant does not limit the present invention, but is anionic such as fatty acid sodium, monoalkyl sulfate, alkyl polyoxyethylene sulfate, alkylbenzene sulfonate, monoalkyl phosphate and the like. Surfactants, cationic surfactants such as alkyltrimethylammonium salt, dialkyldimethylammonium salt, alkylbenzyldimethylammonium salt, amphoteric surfactants such as alkyldimethylamine oxide and alkylcarboxybetaine, polyoxyethylene alkyl ethers, fatty acid sorbitan Nonionic surfactants such as esters, alkylpolyglucosides, fatty acid diethanolamides, alkylmonoglyceryl ethers; glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, etc. or their hydrogen additives), sphingolin lipids (sphingoeline, etc.) and spingosaccharides. Examples thereof include phospholipids such as lipids, derivatives thereof, and mixtures thereof. Among these, phospholipids and the like, derivatives thereof or mixtures thereof and the like can be mentioned. These may be used alone or in combination of two or more, and may be selected by those skilled in the art according to the purpose of use.

With respect to the agent, the invention is not limited, for example, anti-inflammatory agents, fibronectin, albumin, laminin, blood clots or anticoagulant factors (antithrombin, plasmin, urokinase, streptoxinase, fibrinogen activator, thrombin, etc.), Caliclein, kinin, radikinin antagonists, enzymes that do not act on blood, hormones, growth factors such as bone formation factors and cell growth factors, proteinaceous bone growth factors, blood clots or anticoagulants, anticoagulants, osteoporosis remedies, etc. Can be mentioned. These may be used alone or in combination of two or more, and may be selected by those skilled in the art according to the purpose of use.

The filling does not limit the present invention, and examples thereof include polymers, ceramics, metals, granules of any complex thereof, hydrogels, and dried products thereof. Further, the filling material may hold a drug or the like inside, and the above-mentioned drug is exemplified as the drug. The filler for holding the drug is not limited to the present invention, but is, for example, one or more of polyvinyl alcohol, collagen, gelatin, agar, hyaluronic acid, chitin / chitosan, and polyvinyl acetate. Hydrogels made of the above, dried products thereof, biodegradable polymers such as polylactic acid-based polymers and polyethylene glycol-based polymers, and composites of these with calcium phosphate-based ceramics are preferably exemplified. These may be used alone or in combination of two or more, and may be selected by those skilled in the art according to the purpose of use.

The content of any component in the fiber is not limited as long as the effect of the present invention is not hindered, but when any component is contained, the content of any component in the fiber is preferably 50% by weight or less, and the surfactant is preferably 50% by weight or less. 0.05 to 5% by weight is preferably exemplified.

In the present invention, the average fiber diameter of the fibers of the fiber molded product is not limited as long as the effect of the present invention can be obtained, but is preferably 0.05 to 30 μm from the viewpoint of the strength and surface area of the fiber molded product. Here, the fiber diameter means the diameter of the fiber cross section, and is obtained from the average value of at least three points measured by an electron beam microscope image or a confocal laser scanning microscope image of the fiber. When the fiber cross section is elliptical, the average of the length in the major axis direction and the length in the minor axis direction of the ellipse is calculated as the fiber diameter. When the fiber cross section is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or an ellipse.

The fiber-molded object is manufactured by an electrospinning method. The electrospinning method is a known method, in which a polymer solution discharged from a discharge part such as a syringe becomes charged by applying a high voltage, and micro-level or nano-level fibers are attached to a collector. A three-dimensional fiber structure can be manufactured by molding the obtained fiber.

In the present invention, a fiber containing the biocompatible polymer may be produced and a fiber structure may be formed by discharging a solution containing the biocompatible polymer from a discharge portion using an electrospinning method. Here, in producing fibers from a biocompatible polymer, the fibers used in the present invention are not limited as long as they can be produced, but 500 to 5000 V / cm is exemplified as an electric field used for electrospinning. The fiber molded product, that is, the carrier can be produced by laminating, compressing, joining and / or cutting the obtained fibers as necessary. The size and thickness of the carrier may be appropriately set by those skilled in the art according to the purpose of use, the application site, and the like. Although not limiting the present invention, the thickness of the carrier is exemplified by 0.05 to 500 mm. Although not limited to this limitation, from the viewpoint of facilitating the production of the carrier, the thickness of the carrier is preferably 0.5 to 100 mm, more preferably 0.5 to 50 mm. The thickness of the carrier is calculated by directly measuring the height of the fiber molded product without applying pressure to the carrier using a ruler or caliper, or by measuring and calculating the outer shape obtained from a photographed image or the like by image analysis. Obtained by. In the present invention, the thickness means an average value of three or more points of the values measured in this way. In the present invention, the thickness usually refers to the shortest side of the three sides (length, width, height) constituting the carrier.

The solution containing the biocompatible polymer is not limited as long as the desired fiber can be produced, but a mixed solution of the biocompatible polymer and a solvent capable of dissolving the biocompatible polymer, and any of the above-mentioned components are required. A mixed solution further contained is exemplified accordingly. The solvent is also not limited as long as the desired fiber can be produced, and examples of the solvent include chloroform, dichloromethane, N, N-dimethylformamide, formamide, water, formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, trifluoroethanol, 1, Examples thereof include 1,1,3,3,3-hexafluoro-2-propanol and acetone. These may be used individually by 1 type, and may be used in combination of 2 or more type.

In the solution containing the polymer (polymer solution) used for producing the carrier of the present invention, the content of the biocompatible polymer is 2% by weight or more, more preferably 2.5 to 25% by weight, still more preferably 5. To 15% by weight is exemplified. The solvent and arbitrary components in the solution may be appropriately set by those skilled in the art according to the target fiber molded product.

Further, the carrier of the present invention may have holes that penetrate the carrier and / or holes that do not penetrate the carrier, if necessary. The pore size may be appropriately determined according to the purpose. For example, from the viewpoint of more efficiently permeating cells into the carrier, pores having an average pore diameter larger than 10 μm are exemplified, and more preferably 1000 μm larger than the average pore diameter of 10 μm. The following is exemplified. The method for forming the holes is not particularly limited, and those skilled in the art may appropriately form the holes. For example, perforation is exemplified, and more specifically, perforation using a processing laser is exemplified. The same applies when the carrier of the present invention includes a coating layer and / or a high-density layer described later. Here, the average hole diameter is a value calculated as the diameter of a circle having the same area by measuring the projected area of the hole by image analysis or the like.

The carrier of the present invention may have a coating layer on at least a part of the fiber molded product. Although not limiting the present invention, FIG. 4 briefly illustrates a model in which the carrier of the present invention includes a coating layer. The coating layer bridges between fibers on the surface of at least a part of the fiber molded product.

The thickness of the coating layer is not limited as long as the effect of the present invention can be obtained, but is preferably 1000 μm or less, more preferably 0.5 to 10 μm, and further preferably 1 to 5 μm. Here, the thickness of the coating layer is a value obtained by averaging the heights of the coating layers, and is calculated by image analysis of a microscopic image of a cross section or the like. The height of the coating layer refers to a direction in the coating layer that is substantially perpendicular to the fiber molded product. If the coating layer is thin and difficult to measure, it is calculated by forming a coating layer on a smooth metal substrate and then analyzing it with a profilometer or a confocal laser scanning microscope. Even when the fiber molded product has a coating layer in the carrier of the present invention, it is preferable that the ^{bulk density including the coating layer satisfies 0.0001 to 0.25 g / cm 3.} Further, even when the fiber molded product has a coating layer in the carrier of the present invention, it is preferable that the thickness of the carrier including the coating layer is 0.05 to 500 mm.

Further, the coating layer preferably has pores having an average pore diameter of 10 μm or more penetrating the coating layer, and more preferably an average pore diameter of 10 to 1000 μm is exemplified. Here, the average pore diameter is a value calculated as the diameter of a circle having the same area by measuring the projected area of the through hole of the coating layer by image analysis or the like.

Further, it is exemplified that the coating layer preferably has an opening of 50% or more of the surface area of the coating layer. Here, the opening refers to a portion that is present in the coating layer but penetrates the coating layer and the coated fiber molded product is exposed. The surface area of the coating layer is the surface area of the outer shape of the carrier covered by the coating layer, and the surface area of the outer shape of the carrier is imaged from an image obtained by directly measuring the dimensions of the carrier using a ruler or a caliper. It may be calculated by analysis.

By providing the coating layer on the fiber molded product, the shape of the fiber molded product can be maintained more stably. In particular, the lower the bulk density of the fiber molded product, the less stable the fiber molded product tends to be, and by providing the coating layer, the shape of the fiber molded product can be maintained more stably, whereby the alveolar bone in the fiber molded product. It becomes possible to efficiently support the derived undifferentiated osteoblasts, and in particular, it becomes possible to efficiently introduce the cells into the fiber molded product. From this point of view, the fiber molding more preferably comprises a coating layer. From the viewpoint of further enhancing the stability, the coating layer more preferably has a thickness of 1000 μm or less, and the average pore diameter of the holes penetrating the coating layer is 10 μm or more, more preferably 0.5 to 10 μm. And the average pore diameter of the pores penetrating the coating layer is 10 to 1000 μm. Also, from this point of view, the coating layer is preferably composed of agglomerates of fibers. An example of a coating layer having such characteristics is shown in FIG. FIG. 5 is an enlarged photograph of the surface of the coating layer.

The method of forming the coating layer on the fiber molded product is not limited as long as the coating layer can be obtained, but the fibers on the outermost surface thereof are fused to each other on a part or all of the outer surface of the fiber molded body by using a laser or the like. An example is a sealing treatment by allowing the fiber to be formed, whereby a coating layer composed of a fiber layer fused to the fiber molded product can be formed.

Further, when the coating layer is made of agglomerates of fibers, a method of forming a non-woven fiber-forming body on the surface of the carrier by electrospinning is exemplified. In the electrospinning method, a coating layer is formed on the surface of the carrier by placing the carrier between the discharge portion of the polymer solution such as a syringe to which a high voltage is applied and the counter electrode for collecting the fibers. In order to facilitate the formation of the coating layer, the carrier may be irradiated with an ionic wind having a polarity opposite to the polarity applied to the discharge portion of the polymer solution. Further, as a preferred example, from the viewpoint of maintaining the three-dimensional shape of the carrier more stably, in the fibers of the fiber molded body constituting the carrier, a cotton-like fiber in which hydrophilic polymer fibers are formed inside the hydrophobic polymer fibers. When the composite fibers of the above are formed by electrospinning, a coating layer made of non-woven composite fibers can be obtained by switching from the polymer solution to a polymer solution from which the hydrophilic polymer component has been removed, or by appropriately changing the electric field conditions of electrospinning. Can be formed on the outermost surface of the cotton-like fiber molded body, whereby a coating layer can be provided on the surface of the fiber molded body.

The average fiber diameter of the fibers constituting the coating layer is not limited as long as the effects of the present invention can be obtained, but is preferably 0.05 to 30 μm. The fiber diameter is described in the same manner as described above.

Further, the carrier of the present invention may have one or more porous high-density layers in at least a part of the fiber molded product. In other words, the carrier of the present invention may have a layer having a low bulk density (low density layer) and a layer having a high bulk density (high density layer) in the fiber molded product, and in this case, the carrier of the present invention. Can be said to have a coarse and dense structure.

The bulk density of each of the low density layer and the high density layer is in the range of ^{0.0001 to 0.25 g / cm 3 of the bulk density.} Although not limiting the present invention, in the carrier of the present invention, preferably, the bulk density of the high-density layer has a bulk density that is twice or more larger than the bulk density of the low-density layer adjacent to the high-density layer. It has a bulk density 5 times or more more preferably. The high-density layer may be present on at least a part of the surface of the fiber molded product, may be present on at least a part of the inside, or may be present on both of them.

In the carrier of the present invention, even when the fiber molded product has a high-density layer, the bulk density including the high-density layer and the low-density layer may satisfy ^{0.0001 to 0.25 g / cm 3.} preferable. Further, in the carrier of the present invention, even when the fiber molded product has the coating layer, the high density layer and the low density layer, the bulk density including the coating layer and the high density layer is 0.0001 to 0. It is preferable to satisfy 25 g / cm ^3.

The thickness of the high-density layer is not limited, but is preferably 1000 μm or less, and more preferably 5 to 30 μm. As a matter of course, the thickness of the high-density layer and the thickness of the low-density layer do not exceed the thickness of the fiber molded product. Further, even when the fiber molded product has a high-density layer in the carrier of the present invention, it is preferable that the thickness of the carrier including the high-density layer is 0.05 to 500 mm.

From the viewpoint of efficiently proliferating and / or differentiating undifferentiated osteoblasts derived from alveolar bone, the high-density layer consists of an aggregate of fibers.

Further, the high-density layer may be perforated with pores having an average pore diameter larger than 10 μm to form a passage through which cells pass. The method for forming the holes is not particularly limited, but drilling using a processing laser is exemplified. The average pore size is measured in the same manner as described above.

In the present invention, having a high-density layer in the fibrous molded product is useful in that undifferentiated osteoblasts derived from alveolar bone are more efficiently proliferated and differentiated in the high-density layer.

The method of forming the high-density layer on the fiber molded product is not limited as long as the high-density layer can be obtained. When the high-density layer is composed of agglomerates of fibers, a method of forming a non-woven fiber-forming body by electrospinning on the surface of the fiber molded product is exemplified. In the electrospinning method, a high-density layer is formed on the surface of the fiber molded product by placing the fiber molded product between the discharge portion of the polymer solution such as a syringe to which a high voltage is applied and the counter electrode for collecting the fibers. In order to facilitate the formation of the high-density layer, the carrier may be irradiated with an ionic wind having a polarity opposite to the polarity applied to the discharge portion of the polymer solution. Further, as a preferable example, from the viewpoint of maintaining the three-dimensional shape of the carrier more stably, in the fibers of the fiber molded body constituting the carrier, a cotton-like fiber in which hydrophilic polymer fibers are formed inside the hydrophobic polymer fibers. When the composite fibers of the above are formed by electrospinning, a high density of non-woven composite fibers can be obtained by switching from the polymer solution to a polymer solution from which the hydrophilic polymer component has been removed, or by changing the electrospinning electric field conditions. The layer can be formed on the outermost surface of the cotton-like polymer.

The method for forming the high-density layer inside the fiber-molded product is not limited as long as the high-density layer can be obtained, but from the viewpoint of maintaining the three-dimensional shape of the carrier more stably, the fiber-molded body constituting the carrier In the fiber, a cotton-like composite fiber molded body in which the hydrophilic polymer fiber is formed inside the hydrophobic polymer fiber is formed by electrospinning of a polymer solution in which a hydrophobic polymer and a hydrophilic polymer component are mixed. If so, a high-density layer made of non-woven composite fibers can be formed on the outermost surface of the cotton-like molded body (low-density layer) by switching from the polymer solution to the polymer solution from which the hydrophilic polymer component has been removed. it can. Subsequently, by switching to a polymer solution in which a hydrophobic polymer and a hydrophilic polymer component are mixed, cotton-like composite fibers can be formed on the high-density layer.

Another method of forming a high-density layer inside the fiber molding is to electrospin a polymer solution in which a hydrophobic polymer and a hydrophilic polymer component are mixed to form a hydrophilic polymer inside the fibers of the hydrophobic polymer. When a cotton-like polymer molded body in which fibers are formed is formed, a high-density layer made of a non-woven composite fiber is formed by increasing the electric field of electrospinning (low-density layer). ) Can be formed on the outermost surface. By subsequently restoring the electric field, cotton-like composite fibers can be formed on the high-density layer.

By performing the operation a plurality of times, it is possible to form a carrier having a coarse and dense structure in which a plurality of high-density layers are formed at an arbitrary thickness in the above-mentioned range inside the cotton-like molded body. The thickness of the high-density layer and the low-density layer can be easily controlled by, for example, changing the time for accumulating the layers by electrospinning. In addition, by forming a carrier by performing electrospinning while switching between a polymer solution having a different polymer composition and a polymer solution containing a different arbitrary component, drug removal, different cellular responses, and different cell responses are different for each layer inside the carrier. Functions such as biodegradation rate can be added.

The method of forming the high-density layer on both the inside and the surface of the fiber molded product is not limited to the present invention, but for example, a method of forming the high-density layer inside the fiber molded product and fiber molding. A method of forming a high-density layer on the surface of an object may be combined.

The average fiber diameter of the fibers constituting the high-density layer is not limited as long as the effects of the present invention can be obtained, but is preferably 0.05 to 30 μm. The fiber diameter is described in the same manner as described above.

In the present invention, the carrier is not limited to this extent, but the complex is constructed from the viewpoint of more efficiently inducing the differentiation of the supported alveolar bone-derived undifferentiated osteoblasts into osteoblasts in the complex described later. More preferably, in the evaluation using alkaline phosphatase staining shown in Examples described later as an index, the ^{alveolar process is more preferable than the case where carrier 2 (fiber-forming body having a bulk density of 0.21 g / cm 3} and a thickness of about 80 μm) is used. Examples of carriers are capable of efficiently inducing the differentiation of undifferentiated bone-derived osteoblasts into osteoblasts (promoting the induction of differentiation).

Here, the evaluation using the alkaline phosphatase staining as an index shown in the examples described later follows the following procedure. That is, the complex of undifferentiated osteoblasts and the carrier was present in MF medium (manufactured by Toyo Boseki Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone at 37 ° C. and 5% CO2. The cells are cultured underneath, fixed with 10 wt% formaldehyde for 30 minutes, stained with an alkaline phosphatase stain for 5 minutes, and the alkaline phosphatase activity is measured. Efficient induction of differentiation into osteoblasts means that the time for alkali phosphatase positivity is earlier than that when carrier 2 is used as a carrier constituting the complex.

Further, in the present invention, the carrier is not limited to this extent, but from the viewpoint of more efficiently inducing calcification in the complex, the carrier constituting the complex is preferably used as an index using Alizarin red staining shown in Examples described later. In the evaluation of calcification, a carrier capable of inducing calcification (promoting calcification) more efficiently than the case of using the carrier 2 is exemplified.

Here, the evaluation using the alizarin red staining as an index shown in the examples described later follows the following procedure. That is, the complex of undifferentiated osteoblasts and the carrier was present in MF medium (manufactured by Toyobo Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone at 37 ° C. and 5% CO _2. Incubate under, fix in 10 wt% formaldehyde for 30 minutes and stain with alizarin red for 5 minutes. Efficient calcification means that the calcification time is faster than when the carrier 2 is used as the carrier constituting the complex in the dyeing.

The form, size, and the like of the carrier thus obtained are not particularly limited, and may be appropriately designed according to the damaged site of the bone tissue to be applied.

Although not limiting the present invention, model diagrams of the carriers obtained as described above are illustrated in FIGS. 6 to 11.

Complex In the present invention, the complex of alveolar bone-derived undifferentiated osteoblasts and a carrier is limited as long as the alveolar bone-derived undifferentiated osteoblasts are supported on at least a part of the carrier. Not done.

The carrying method is not limited as long as the cells come into contact with the carrier, and for example, the carrier is impregnated, coated, sprayed, or injected with a solution (including liquid or semi-solid) and / or a solid substance containing the cells. , Implantation and the like, and the cell may be in the state of a cell spheroid. Further, for example, a method of culturing while covering at least a part of the carrier with a cell sheet or a sheet-like material carrying the cells to attach the cells to the carrier can be mentioned.

The amount of the cells carried on the carrier is also not limited as long as the effect of the present invention can be obtained. For example, undifferentiated osteoblasts derived from the alveolar bone are preferably 1 × 10 ³ to ^{1 cm 3 per 1 cm 3 of the carrier.} Examples of supported amounts are 5 × 10 ⁶ cells, more preferably 1 × 10 ⁴ to 4 × 10 ^{6 cells.}

Although the present invention is not limited, for example, by temporarily holding a solution or the like containing the cells in the fiber molded body, and then discharging the solution such as the cell culture solution held inside by compressing the fiber molded body, the solution or the like is discharged. Cells can be easily concentrated in the carrier. The density of the fiber molded product after compression may be appropriately used as the composite of the present invention as a preferable range for use as a carrier.

According to the composite of the present invention, since excellent bone tissue regeneration ability is possible, it can be used to regenerate bone tissue and restore it to a normal state in a disease associated with bone tissue damage. The composite of the present invention can be applied to all types of bone tissue damage such as alveolar bone damaged by periodontal disease, bone damaged by osteosarcoma, bone damaged by bone metastatic cancer, and fractures. The target is not limited. In the complex of the present invention, the alveolar bone damaged by periodontal disease is a suitable application target from the viewpoint of exerting a remarkably excellent therapeutic (regeneration) effect.

The complex of the present invention is used by administering (transplanting) to a damaged site of bone tissue. In addition, the complex of the present invention may be directly administered (transplanted) to the damaged site of bone tissue, and after at least a part of the undifferentiated osteoblasts supported in vitro is grown on a carrier, the bone It may be administered (transplanted) to the damaged site of the tissue. In addition, the complex of the present invention may be administered (implanted) to a damaged site of bone tissue after differentiating at least a part of the undifferentiated osteoblasts into osteoblasts in vitro. From this, the present invention also provides a complex in which at least a part of the undifferentiated osteoblasts derived from alveolar bone is differentiated into osteoblasts derived from alveolar bone, which is an osteoblast. It can also be called a cell differentiation complex. In addition, the complex of the present invention may be administered (transplanted) to the damaged site of the bone tissue after the bone tissue is formed on at least a part of the carrier in vitro. From this, the present invention also provides a complex in which at least a part of the undifferentiated osteoblasts derived from the alveolar bone is in a calcified state, which can also be said to be a calcified complex.

As a method for administering (transplanting) the complex of the present invention to a damaged site of bone tissue, a method conventionally known in the art may be followed, depending on the type of target bone tissue, the degree of damage, and the like. It may be set appropriately.

In the regeneration of bone tissue in the present invention, the dose of the complex of the present invention may be appropriately set according to the degree of disease symptoms, the sex and age of the patient, and the like. , The applied amount of the undifferentiated osteoblasts derived from alveolar bone is 1 × 10 ⁵ to 1 × 10 ⁷ cells, preferably 2 × 10 ⁶ to 4 × 10 ⁶ cells, more preferably 2.5 × 10 ^{6 to} 3. It may be set to be .5 × 10 ^{6 cells.}

The complex of the present invention may be used as a complex for autologous transplantation or as a complex for allogeneic transplantation. From the viewpoint of further suppressing rejection, the complex of the present invention is preferably used for autologous transplantation.

By administering (transplanting) the complex of the present invention in this way, the engraftment rate of undifferentiated osteoblasts derived from alveolar bone at the damaged site of bone tissue is further increased, which is clinically useful, which has been difficult in the past. Bone tissue regeneration can be further promoted.

The complex of the present invention may, if desired, coexist with a further pharmaceutically acceptable dilution carrier. Here, examples of the pharmaceutically acceptable dilution carrier include a buffer solution such as physiological saline. The complex of the present invention may further coexist with a pharmacologically active ingredient, if necessary. From these facts, the complex of the present invention can be used as a cell preparation for bone tissue regeneration. In addition, the composite of the present invention may be used together with arbitrary components such as a pharmaceutically acceptable buffer solution and a pharmacologically active ingredient, if necessary, and these arbitrary components may be appropriately selected by those skilled in the art. ..

In addition, the composite of the present invention may be further used in combination with a conventionally known scaffold as long as it does not interfere with the effects of the present invention and is pharmaceutically acceptable, as such a scaffold. For example, a gel-like or porous material having biocompatibility can be mentioned. Specific examples of such scaffolds include fibrin gel (fibrin glue), hydroshikiapatite, PGLA (poly DL-lactic-co-glycolic acid) -collagen sponge and the like. The complex of the present invention may be administered (implanted) to the damaged site of bone tissue together with such a scaffold.

From this, it can be said that the present invention provides a cell preparation for bone tissue regeneration containing the complex. The cell preparation may contain any of the above-mentioned components, if necessary, and may contain the above-mentioned conventionally known scaffolds. Those skilled in the art can determine the selection of each component and scaffold, its content, and the like. It may be selected as appropriate.

Bone Tissue Regeneration Method Using The Complex The present invention also provides a method for treating bone tissue damage, which comprises a step of administering the complex to the bone tissue site of a patient with bone tissue damage. .. In the treatment method, the damage to the bone tissue to be treated, the complex used, the dose of the complex, the administration method and the like are as described above.

By administering (transplanting) the complex of the present invention in this way, the engraftment rate of undifferentiated osteoblasts derived from alveolar bone at the damaged site of bone tissue is further increased, and bone tissue regeneration, which has been difficult in the past, is further enhanced. Can be further promoted.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
Example 1: Preparation of undifferentiated osteoblasts derived from alveolar bone Four patients (66 years old, 53 years old, 52 years old, 27 years old) who obtained informed outlets based on the rules of the Ethics Committee. Using the alveolar bone removed from the patient) at the time of tooth extraction, undifferentiated osteoblasts derived from the alveolar bone were prepared.

The obtained alveolar bone was placed in 4 ml of PBS (Phosphate buffered saline, pH 7.2) containing 2 mg / ml bacterial collagenase (> 1.5 U / mg; Collagenase P, manufactured by Roche) at 37 ° C. The enzymatic reaction was carried out for 20 minutes. After the reaction, the cells released by centrifugation were collected after adding an equal amount of bovine serum to the enzyme solution, and the remaining alveolar bone was treated with the enzyme again under the same conditions as described above. This operation was repeated, and finally 8 times of enzyme treatment was performed. Of the 8 cell fractions recovered after each enzyme treatment, the cell fractions recovered after the first and second enzyme treatments are discarded, and the remaining 6 cell fractions are used in the MF start medium (Toyobo). , Tokyo, Japan) The cells were placed in a 35 mm culture dish containing 4 ml and cultured under the conditions of _{5% CO 2 and 37 ° C.}

When the cells reached 80% confluence in the culture dish, the cells were released with PBS containing 0.25% trypsin / 1 mM EDTA to recover undifferentiated osteoblasts (HAOB) from human alveolar bone. In the tests shown below, HAOB obtained from cell fractions recovered after the fifth enzyme treatment was used. Since then, HAOB derived from alveolar bone obtained from a 66-year-old patient is HAOB1; HAOB derived from alveolar bone obtained from a 53-year-old patient is HAOB2; HAOB derived from alveolar bone obtained from a 52-year-old patient is used. HAOB3; and HAOB derived from alveolar bone obtained from a 27-year-old patient are referred to as HAOB4, respectively.

Example 2: Evaluation of proliferative ability of undifferentiated osteoblasts derived from alveolar bone The HAOB obtained in Example 1 was placed in MF medium (Toyobo, Tokyo, Japan) so as to have a concentration of 3 × 10 ⁴ cells / ml. The seeds were seeded and subcultured for 70 days by changing the medium every 3 days. During that time, HAOB population doubling (PD) was measured.

In addition, HAOB3 of 35PD was induced in interphase by treatment with Colcemid (Karyo Max; Gibco BRL; 100 ng / ml for 6 h). For HAOB3 (about 50 cells) induced in interphase, the chromosomal structure of HAOB3 was analyzed by the G band method. Furthermore, for HAOB3 thus induced in interphase, the chromosomal structure of HAOB3 was analyzed by the SKY (Spectral Karyotyping) method.

The results are shown in FIG. From the results of A) in FIG. 12, it was confirmed that HAOB (HAOB1 to 3) obtained from the alveolar bone of middle-aged and elderly people showed the same growth rate as HAOB (HAOB4) obtained from the alveolar bone of young people. It was. In addition, from the results of analyzing the chromosomal structure of interphase HAOB3 shown in B) of FIG. 12, a normal diploid image was confirmed, indicating that HAOB has a normal and stable proliferative capacity. It was. Similar results were observed in other HAOBs.

Example 3: Evaluation of Proliferative Characteristics of Undifferentiated Osteoblasts Derived from Alveolar Bone HAOB3 obtained in Example 1 ^{was seeded in a 96-well plate at 2 × 10 4} cells / well, and Dulbecco's modified Eagle's medium without serum was added. After culturing in (DMEM) for 24 hours, the cells were cultured in DMEM medium containing 10 ng / ml bFGF, PDGFAA, PDGFAB or PDGFBB for 96 hours, and the cell proliferation activity was evaluated. As a control, HAOB3 after culturing in DMEM medium was similarly cultured in DMEM or MF medium containing serum-free DMEM and 20% by volume FCS for 96 hours to evaluate cell proliferation activity. The cell proliferation activity was measured using a Celltiter-Glo luminesescent cell viability assay (promega) according to a method specified by the vendor.

The results are shown in FIG. The vertical axis of FIG. 13 shows the relative value of cell proliferation activity calculated with the number of cells when cultured using DMEM without serum added as 1. From this result, it was clarified that the growth of HAOB was remarkably improved when PDGFAA, PDGFAB or PDGFBB was added, and that culturing in the presence of these growth factors was effective for the growth of HAOB. It was.

Example 4: Evaluation of differentiation potential of undifferentiated osteoblasts derived from alveolar bone (In vitro) -1
^{After inoculating 2} ml of HAOB3 obtained in Example 1 in a culture dish at a concentration of 3 × 10 ⁴ cells / ml / cm 2, 100 nM dexamethasone, 50 μg / ml alcorbic acid, 10 mM β-glycerophosphate, and 100 ng / ml. ^{Replace with 1 ml / cm 2} of MF medium containing rhBMP-2 (Recombinant human bone morphogenetic protein-2) (hereinafter referred to as rhBMP-2 added medium), and incubate for 9 days while changing the medium every 3 days. The differentiation characteristics of HAOB3 were evaluated. For comparison, instead of the rhBMP-2-added medium, culture was performed under the same conditions as above using a medium having the same composition as the above rhBMP-2-added medium except that it did not contain rhBMP-2, and the differentiation characteristics of HAOB3. Was evaluated.

Furthermore, for comparison, human epidermis-derived fibroblasts (HFF) (Takara Bio Inc.) were cultured in hBMP-2-added medium or MF medium under the same conditions as above, and the differentiation characteristics of HFF were evaluated.

In this study, the differentiation characteristics were measurement of alkaline phosphatase activity, Alizarin red staining, measurement of expression level of osteoblast differentiation markers (RUNX2, OSTERIX, OSTEOCALCIN (OCN), BONE SIALOPROTEIN (BSP)), and OSTEOPONTIN. It was evaluated by immunostaining with antibodies against (OPN) and OCN. The specific measurement conditions for this are as follows.

<Measurement of alkaline phosphatase activity>
After fixing the cells in 4% paraformaldehyde for 20 minutes, 0.1 mg / ml naphthol AS-MX phosphate (Sigma), 0.5% NN dimethyl formamide (Sigma), 2 mM MgCl2, 0.6 mg / ml Fast Blue BB salt (Sigma) Alkaline phosphatase activity was measured by reacting with 0.1 M TRIS-HCl (pH 8.5) solution containing.

<Alizarin red dyeing>
Cells were fixed with 4% paraformaldehyde for 20 minutes and then stained with 2% alizarin red S (pH 6.4) (Sigma) for detection.

<Measurement of osteoblast differentiation marker expression level>
Total RNA was extracted from the cells and the expression levels of RUNX2, OSTERIX, OCN, and BSP were measured by PCR. Extraction of total RNA from cells was performed using Isogen (Nippon Gene, Tokyo, Japan) according to the method specified by the vendor. cDNA was prepared using 1 μg) of total RNA and reverse transcriptase (M-MLV reverse transcriptase, Invitrogen Corporation). The expression level of mRNA was analyzed by AB 7300 Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems). The sequence of the primers used is as follows.
OSTERIX (Forward primer: CTGAAGAATGGGTGGGGAAGG (SEQ ID NO: 1), reverse primer: GGCCTCTGTCCTCCTAGCTC (SEQ ID NO: 2))
RUNX2 (Forward primer: GAAACTCAACAGATTAACTATCGTTTGC (SEQ ID NO: 3), Reverse primer: GAATTTATCACAGATGGTCCCTAATGG (SEQ ID NO: 4))
OSTEOCALCIN (Forward primer: CACACTCCTCGCCCTATTGG (SEQ ID NO: 5), Reverse primer: TGCACCTTTGCTGGACTCTG (SEQ ID NO: 6))
BONE SIALOPROTEIN (Forward primer: CGAATACACGGGCGTCAATG (SEQ ID NO: 7), Reverse primer: GTAGCTGTACTCATCTTCATAGGC (SEQ ID NO: 8))
BMP2 (Forward primer: CCAGAAACGAGTGGGAAAAC (SEQ ID NO: 9), Reverse primer: AATTCGGTGATGGAAACTGC (SEQ ID NO: 10))
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Forward primer: AAGAGCACAAAGAGGAAGAGAGAGAC (SEQ ID NO: 11), Reverse primer: TTATTGATGGTACATGACAAGGTG (SEQ ID NO: 12)).

<Immunostaining with antibodies against OPN and OCN>
After fixing the cells with 4% paraformaldehyde, immunostaining was performed with an antibody against OPN and an antibody against OCN, and the expression levels of OPN and OCN were measured.

The results are shown in FIGS. 14-1 and 14-2. A) of FIG. 14-1 shows the alizarin red staining result (left) and the measurement result of alkaline phosphatase activity (right). As is clear from the left figure of A) in FIG. 14-1, HAOB3 was highly calcified because the staining with alizarin red was remarkably observed by culturing in the hBMP-2 added medium (+ in the figure). It turned out that the ability was expressed. It was also confirmed that HAOB3 was significantly improved in alkaline phosphatase activity, which is an index of bone formation, by culturing in hBMP-2 added medium, as shown in the right figure of A) in FIG. 14-1. It was. On the other hand, in HFF, even when cultured in hBMP-2 added medium, it was not stained with alizarin red and no alkaline phosphatase activity was observed.

Furthermore, B) of FIG. 14-1 shows the results of measuring the expression levels of RUNX2, OSTERIX, OCN and BSP. In B) of FIG. 14-1, the expression level of each differentiation marker is shown as a relative value with the expression level of β-Actin as 100. From this result, it was confirmed that the expression levels of RUNX2, OSTERIX, OCN and BSP were increased and the bone-forming ability was expressed by culturing HAOB3 in the hBMP-2-added medium. On the other hand, in HFF, the expression of the above differentiation marker was not observed in either the hBMP-2 added medium or the MF medium.

In addition, FIG. 14-2 shows the results of immunostaining using antibodies against OPN and OCN. As can be seen from FIG. 14-2, HAOB3 not stimulated with rhBMP-2 slightly expressed OPN and OCN, but HAOB3 stimulated with rhBMP-2 showed a strongly positive reaction to OPN and OCN antibody. It was. From this result, it was confirmed that HAOB showed the properties of undifferentiated osteoblasts before the induction of differentiation, and differentiated into OPN-positive and OCN-positive osteoblasts by rhBMP-2 stimulation.

From these facts, it was found that HAOB can successfully differentiate from undifferentiated osteoblasts to osteoblasts.

Example 5: Evaluation of differentiation potential of undifferentiated osteoblasts derived from alveolar bone (In vitro) -2
HAOB3 obtained in Example 1 was seeded in MF medium (Toyobo, Tokyo, Japan) so as to have a concentration of 3 × 10 ⁴ cells / ml, and cultured to 29 PD while changing the medium every 3 days. It was. HAOB3 of 6PD, 11PD, 16PD, 21PD, 24PD and 29PD was cultured in rhBMP-2 supplemented medium under the same conditions as in Example 4 above, followed by alizarin red staining and osteoblast differentiation markers (RUNX2, OSTERIX, OCN). , BSP) expression level was measured.

The results are shown in FIG. A) of FIG. 15 shows the result of alizarin red staining, B) shows the result of measuring the expression level of OCN, and C) shows the result of measuring the expression level of RUNX2, OSTERIX and BSP. B) and C) of FIG. 15 show relative values of the expression level of each differentiation marker with the expression level of β-Actin as 100. As is clear from FIG. 15, even with HAOB3 of 16PD, the bone forming ability could be sufficiently expressed and the osteoblast differentiation ability was retained.

From this, it was confirmed that HAOB has sufficient osteoblast differentiation ability and bone formation ability.

Example 6: Evaluation of the ability of undifferentiated osteoblasts derived from alveolar bone to differentiate into bone cells (In vitro) -3
HAOB3 obtained in Example 1 was seeded in a culture dish at a concentration of 3 × 10 ⁴ cells / ml / cm ² , followed by 100 nM dexamethasone, 50 μg / ml alcorbic acid, 10 mM β-glycerophosphate, and 50, 100, The cells were replaced with MF medium containing 200, 500 or 1000 ng / ml rhBMP-2 and cultured for 9 days while changing the medium every 3 days to evaluate the differentiation characteristics of HAOB3. Differentiation characteristics are determined by measuring alkaline phosphatase activity, alizarin red staining, and measuring the expression level of osteoblast differentiation markers (RUNX2, OSTERIX, OCN, BSP) in the same manner as in Example 4 above. evaluated.

For comparison, femur-derived osteoblasts (NHOst) (Lonza Walkersville Inc, MD, USA) were cultured under the same conditions as above, and their differentiation characteristics were evaluated in the same manner.

The results are shown in FIG. A) of FIG. 16 shows the alizarin red staining result (left) and the measurement result of alkaline phosphatase activity (right). From this result, HAOB3 was observed to have similar activity in terms of alkaline phosphatase activity as compared with NHOst, but in terms of calcification ability, alizarin red staining-positive calcification was formed at a low concentration of rhBMP-2. It was confirmed that the bone-forming ability can be strongly expressed.

In addition, B) of FIG. 16 shows the results of measuring the expression levels of HAOB3 and NHOst RUNX2, OSTERIX, and BSP induced to differentiate with 100 ng / ml rhBMP-2. In B) of FIG. 16, the expression level of each differentiation marker is shown as a relative value with the expression level of β-Actin as 100. From this result, it was confirmed that in 100 ng / ml rhBMP-2, HAOB3 was strongly induced to differentiate as compared with NHOst, and was able to strongly express bone-forming ability.

Example 7: Evaluation of differentiation potential of undifferentiated osteoblasts derived from alveolar bone (In vitro) -4
HAOB3 or NHOst was mixed with MF medium containing 100 nM dexamethasone, 50 μg / ml alcorbic acid, 10 mM β-glycerophosphate, and 100 ng / ml rhBMP-2 at a concentration of 3 × 10 ⁴ cells / ml / cm ^2. 0.5 ml was added onto the cover glass. This was cultured for 72 hours under the conditions of 5% CO _{2 and 37 ° C.} Then, the culture supernatant was removed from the cover glass, fixed in 50% acetone / methanol solution for 2 minutes, and then blocked with PBS containing 1 mg / ml bovine albumin. The cells on the cover glass thus blocked are allowed to react with an anti-OCN monoclonal antibody (clone GluOC4-5, Takara, Tokyo, Japan) and then reacted with an anti-mouse alexa 488 secondary antibody (Invitrogen Corporation). This stained OCN. In addition, anti-OSTEOPONTIN (OPN) polyclonal antibody (O-17, IBL, Gunnma, Japan) was allowed to act on the blocked cells on the cover glass, and then reacted with anti-rabbit alexa 555 secondary antibody (Invitrogen Corporation). The OPN was stained by allowing it to stain. Furthermore, the cells on the blocked cover glass were subjected to nuclear staining with DAPI (4', 6-diamino-2-phenylindole).

The cells thus stained were observed with a confocal laser scanning microscope (LSM510; Carl Zeiss MicroImaging, Jena, Germany) using laser beams at 403 nm, 488 nm and 543 nm.

The results are shown in FIG. In FIG. 17, a is the result of staining the OPN of the differentiated HAOB3, b is the result of staining the OCN of the differentiated HAOB3, c is the image of a and b superimposed, and d is the differentiation induction. As a result of staining the OPN of NHOst, e is the result of staining the OCN of NHOst which has been induced to differentiate, and f is an image in which d and e are superimposed. From this result, all of the differentiation-induced HAOB3 showed a positive reaction to the anti-OPN antibody and the anti-OCN antibody, and the expression level of OPN and OCN was also significantly higher than that of NHOst.

Example 8: Evaluation of Differentiation Characteristics of Undifferentiated Osteoblasts Derived from Alveolar Bone After seeding HAOB3 or NHOst obtained in Example 1 at a concentration of ^{3 × 10 4} cells / ml / cm ^{2 in a culture dish, The cells were replaced with 1 ml / cm 2 of} MF medium containing 100 nM dexamethasone, 50 μg / ml alcorbic acid, 10 mM β-glycerophosphate, and 100 ng / ml rhBMP-2, and the cells were cultured for 9 days while changing the medium every 3 days. The expression level of BMP-2 was measured by the Real-Time PCR method in the cells 9 days after culturing. RNA collection from cells and cDNA synthesis were carried out by the same method as in Example 4 above. RT-PCR was performed using Takara EX taq (Takara, Tokyo, Japan) according to the method specified by the vendor.

The results are shown in FIG. As shown in FIG. 18, the cells induced to differentiate from HAOB3 expressed 200 times or more of BMP-2 as compared with the cells induced to differentiate from NHOst. This result also supports that HAOB has characteristics that are clearly different from those of NHOst.

Example 9: Analysis of genes specific to undifferentiated osteoblasts derived from alveolar bone The following tests were performed to analyze the expression of genes specific to undifferentiated osteoblasts derived from alveolar bone.

HAOB1 to 4 obtained in Example 1 were seeded in MF medium (Toyobo, Tokyo, Japan) so as to have a concentration of 3 × 10 ⁴ cells / ml, and cultured to 35 PD while changing the medium every 3 days. Was done.

Then, total RNA was extracted from HAOBs of 6PD, 11PD, 16PD, 21PD, 24PD, 29PD and 35PD, and the intensities of expressed genes in HAOBs of 6PD and 35PD were analyzed. The intensity of the expressed gene is determined by using the HT Human Genome U133 Array Plate Set (Gene Chip; Affymetrix, CA, USA) containing a full-length probe of about 3,3000 genes, and the Affymetrix manual [http: // www. I followed .affymetrix.com/support/technical/index.affx]. Data analysis was performed using the Gene Chip Operation System (Affymetrix CA, U.S.A.) and GeneSpringGX software (Silicon Genetics). In order to standardize the change in stainability between chips, the average of the median measurements of all genes on the chips was adopted. In addition, eigenvectors for raw data and logarithmically transformed data were prepared by principal component analysis (PCA method) using singular value decomposition (SVD). (References: Kami D, Shiojima I, Makino H, Matsumoto K, Takahashi Y, Ishii R, Naito AT, Toyota M, Saito H, Watanabe M, Komuro I, Umezawa A: Gremlin enhances the determined path to cardiomyogenesis, PLoS ONE , 3: e2407.2008)

For comparison, human epidermis-derived fibroblasts (HFF) (Takara Bio Co., Ltd.), human osteosarcoma cells (MG63) (RIKEN BioResource Center), and femur-derived osteoblasts (NHOst) (Lonza Walkersville Inc.) , MD, USA), the intensity of the expressed gene was analyzed in the same manner.

As a result of PCA analysis, it was found that there is a group of genes whose expression decreases as PD increases. Among these genes, genes highly expressed in HAOB were screened, and the expression level of the genes was measured by real-time PCR. The result is shown in A) of FIG. In A) of FIG. 19, the expression level of each gene is shown as a relative value with the expression level of β-Actin as 100. As shown in A) of FIG. 19, it was confirmed that STMN2, NEBL, and MGP were highly expressed in HAOB of 6PD, but hardly expressed in HAOB3 of 35PD.

In 6PD of HAOB3, the expression of these genes and the gene expression levels of the bone marker genes RUNX2, OSTERIX, OSTEOCALCIN and BSP were measured by real-time PCR. The result is shown in B) of FIG. In B) of FIG. 19, the expression level of each gene is shown as a relative value with the expression level of β-Actin as 100. As shown in B) of FIG. 19, the expression levels of STMN2, NEBL and MGP were clearly high. Moreover, as shown in C) of FIG. 19, as a result of analyzing the gene change rate of HAOB3 in 6PD and 35PD, OCN showed the highest change rate. However, since the expression level of OCN in 6PD is low, it is considered that it is not suitable as a marker for distinguishing HAOB.

Next, the expression levels of STMN2, NEBL and MGP in HAOB1-4 of human epidermis-derived fibroblasts (HFF), human osteosarcoma cells (MG63), femur-derived osteoblasts (NHOst) and PD6 were measured by real-time PCR. did. The primer sequences used in real-time PCR are as follows.

STMN2 (Forward primer: acgtctgcaggaaaaggaga (SEQ ID NO: 13), Reverse primer: acgatgtagtcgccgtctct (SEQ ID NO: 14))
NEBL (Forward primer: cattcccaaggctatggcta (SEQ ID NO: 15), Reverse primer: acgatgtagtcgccgtctct (SEQ ID NO: 16))
MGP (Forward primer: cgcttcctgaagtagcgatt (SEQ ID NO: 17), Reverse primer: ccctcagcagagatggagag (SEQ ID NO: 18))
The obtained results are shown in FIG. FIG. 20 shows relative values of the expression level of each gene with the expression level of β-Actin as 100. As can be seen from FIG. 20, clearly high expression of STMN2, NEBL and MGP was observed in HAOB.

From the above results, it was confirmed that the expression of STMN2, NEBL and MGP can be a specific marker for HAOB.

Example 10: Preparation of carrier
Example 10-1
The carrier was produced according to the following procedure. After dissolving poly-L-lactic acid (PLLA) (trade name: RESIMER L206s, manufactured by Evonik Rohm GmbH) in chloroform to a concentration of 10 wt%, the resulting solution contains 0.5 to 13 wt% by weight based on PLLA. A formamide solution in which gelatin (trade name: Medigelatin, manufactured by Nippi Co., Ltd.) was dissolved was added in an amount of 5 to 25 vol% and mixed to prepare a polymer solution. While discharging the prepared polymer solution from a syringe, an electric field of 1000 V / cm was applied to spin the polymer solution by electrospinning, and the polymer solution was collected on a collector (counter electrode) to form a cotton-like molded product.

When the amount of gelatin added to the formamide solution was less than 5 vol% and the amount of gelatin added was less than 1.3 wt%, a cotton-like molded product could not be obtained. Further, if the amount of the formamide solution of gelatin added is too large, the viscosity of the polymer solution obtained by mixing becomes too high, and it becomes difficult to perform electrospinning. When the amount of gelatin in the formamide solution added was about 25 vol% or less under the above conditions, electrospinning could be carried out satisfactorily, and a cotton-like formed product could be produced.

Next, change the electric field conditions from 1000 V / cm to 2000 V / cm without changing the composition of the polymer solution, or change the polymer solution to chloroform with 10 wt% PLLA without changing the electric field conditions. After dissolving so as to be, N, N-dimethylformamide was added so as to be 1.1 vol%, and the mixture was switched to a polymer solution obtained by mixing, and electrospinning was performed on the obtained cotton-like molded product. As a result, a coating layer (shape stabilizing layer) was formed on the cotton-like polymer. A coating layer was formed on the surface (upper surface and lower surface) of the cotton-like molded product so that the thickness of the coating layer was 2 to 3 μm. The cotton-like fiber molded body coated with the coating layer ^{was adjusted to have a bulk density of about 0.003 g / cm 3} and subjected to molding treatment to prepare a cell culture carrier for regenerative medicine. This was designated as carrier 1 (cotton-like carrier). A model diagram of carrier 1 is shown in FIG.

As for the bulk density, the fiber molded body to which the coating layer after the molding process is attached is substantially a rectangular parallelepiped as a whole, and the volume is calculated by measuring the length, width, and thickness thereof, and the ultra-micro balance is used. It was calculated by weighing the weight and dividing the weight by the volume.
Example 10-2
In Example 10-1, after dissolving PLLA in chloroform to a concentration of 10 wt%, an N, N-dimethylformamide solution in which gelatin was dissolved was added so as to a concentration of N, N-dimethylformamide to 1.1 vol%. By spinning and collecting in the same manner except that a mixed polymer solution was used, fibers were formed into a cloth shape to form a fiber-forming body having a predetermined thickness (about 80 μm), and the bulk density of the fiber-forming body was obtained. Was adjusted to 0.21 g / cm ^3. This was designated as carrier 2 (nonwoven fabric-like carrier). The bulk density of carrier 2 was calculated in the same manner as described above.

Example 10-3
After dissolving PLLA in chloroform to a concentration of 10 wt% in Example 10-1, the electrospinning conditions were changed to an electric field of 2000 V / cm, and the fibers were spun and collected in the same manner to form a cloth-like fiber. It was formed to form a fiber-forming body having a predetermined thickness (about 80 μm), and the bulk density of the fiber-forming body was adjusted to ^{0.21 g / cm 3.} This was designated as carrier 3 (nonwoven fabric-like carrier). The bulk density of the carrier 3 was calculated in the same manner as described above.

Example 10-4
While forming the cotton-like molded product (low density layer) by the same procedure as in Example 10-1, the N, N-dimethylformamide used in Example 10-2 was reduced to 1.1 vol% of the polymer solution. A cloth-like high-density layer was formed by switching to the polymer solution added and mixed so as to be, and spinning and collecting in the same manner as in Example 10-2 (the thickness of the high-density layer is 5 to 20 μm). Then, by switching to the polymer solution used in Example 10-1 and spinning and collecting in the same manner as in Example 10-1, a structure having a coarse and dense structure of a cotton-like molded body and a cloth-like formed body. Formed a body. After forming a coating layer on the obtained structure in the same manner as in Example 10-1, the bulk density ^{was adjusted to 0.0035 g / cm 3} and molding treatment was performed to obtain a carrier. This was designated as carrier 4 (coarse and dense structure carrier). The bulk density of the high-density layer after adjustment was 0.21 g / cm ³ , and the bulk density of the low-density layer was 0.003 g / cm ³ .

Further, by laminating the polymer solution used in Example 10-1 and the polymer solution used in Example 10-2 while alternately switching, the molded product contains a plurality of low-density layers and high-density layers. It was also possible to form a carrier with a structure. For example, three high-density layers (thickness of the high-density layer is 5 to 20 μm) are formed to form a coating layer in the same manner as in Example 10-1, and then a low-density layer having a height of 5 mm. By performing the molding process together with this, it was possible to produce a carrier having ^{a bulk density of 0.0043 g / cm 3} in which high-density layers were formed at intervals of about 1.25 mm. The bulk density of the adjusted high-density layer and low-density layer was in the range of ^{0.0001 to 0.25 g / cm 3 as described above.}

In this case, the fibers constituting the cotton-like molded body (low density layer) form fibers containing a hydrophilic polymer having an average diameter of 0.5 to 20 μm inside the fibers containing a hydrophobic polymer having an average diameter of 0.5 to 20 μm. At least a part of the surface of the fiber containing the hydrophobic polymer was coated with the hydrophilic polymer, and the high density layer was not in such a state.

Example 10-5
While forming a cotton-like molded body (low density layer) in the same procedure as in Example 10-1, the electrospinning condition was changed from 1000 V / cm to an electric field of 2000 V / cm, and spinning and trapping were performed in the same manner. A cloth-like high-density layer is formed by collecting, and then the condition is changed to 1000 V / cm again, and the cotton-like molded body (low-density layer) and cloth-like are formed by spinning and collecting in the same manner. A structure having a coarse and dense structure with the body (high density layer) was formed. After forming a coating layer on the obtained structure in the same manner as in Example 10-1, the bulk density ^{was adjusted to 0.0035 g / cm 3} and molding treatment was performed to obtain a carrier having a thickness of about 10 mm. This was designated as carrier 5 (coarse and dense structure carrier). The bulk density of the adjusted high-density layer and low-density layer was in the range of ^{0.0001 to 0.25 g / cm 3 as described above.}

It was also possible to form a carrier having a plurality of coarse and dense structures inside the compact by laminating while alternately switching the electrospinning conditions to an electric field of 1000 V / cm and 2000 V / cm. For example, three high-density layers (thickness of the high-density layer is 0.5 to 20 μm) are formed to form a coating layer in the same manner as in Example 10-1, and then together with the low-density layer so that the height becomes 5 mm. By performing the molding process, it was possible to produce a carrier having ^{a bulk density of 0.0043 g / cm 3} in which high-density layers were formed at intervals of about 1.25 mm. The bulk density of the adjusted high-density layer and low-density layer was in the range of ^{0.0001 to 0.25 g / cm 3 as described above.}

In this case, the fiber containing the hydrophilic polymer having an average diameter of 10 to 1000 nm is formed inside the fiber containing the hydrophobic polymer having an average diameter of 0.5 to 20 μm, and at least a part of the surface of the fiber containing the hydrophobic polymer is formed. The fibers coated with the hydrophilic polymer were formed on the entire molded body including the high-density layer.

Example 11: Cell culture on a carrier
Cell culture The above-mentioned carrier 5 (Example 10-5, coarse-dense structure carrier) ^{was cut into 1 × 1 cm 2} (that is, length 1 cm, width 1 cm, height (thickness) about 1 cm) and immersed in 70 wt% ethanol for sterilization. After the treatment, the cells were allowed to stand in each well of a 24-well plate, immediately washed with PBS (-), and replaced with MF medium. Thereafter, the HAOB3 1 in × 10 ⁵ cells / carrier (to ie cut upper carrier) were seeded, CO ₂ incubator and incubated for 14 days under conditions of 37 ° C. in, WST8 method (tetrazolium salt WST8 intracellular dehydration It is reduced by a prime enzyme to produce water-soluble formazan. Since the number of cells and the amount of formazan produced are in a linear proportional relationship, the number of living cells can be determined by directly measuring the 450 nm absorbance of this formazan. The absorbance was measured over time using VERSAMax (manufactured by Molecular devices), and cell proliferation was examined. The experiment was carried out in the same manner except that the carrier 2 (Example 10-2, that is, 1 × 1 cm ^{2 in length and width and about 80 μm in height) was used instead of the carrier 5.} Further, as a comparative example, HAOB was seeded and incubated in the same manner as in the carrier 5 except that a collagen sponge (trade name: collagen sponge, manufactured by Nitta Gelatin Co., Ltd.) used as a carrier in conventional regenerative medicine was used.

Results The results are shown in FIG. As is clear from FIG. 21, cell proliferation in the collagen sponge resulted in remarkable results 1 to 4 days after culturing. However, after the 7th day of culturing, a remarkable proliferative tendency was observed on the carrier 5, and after 14 days of culturing, remarkable cell proliferation was observed on the carrier 5, while no significant cell proliferation was observed on the collagen sponge. With carrier 2, cell proliferation was remarkable 1 to 4 days after culturing, and remarkable cell proliferation was also observed 14 days after culturing.

From this, it was confirmed that the carrier 5 is a carrier suitable for the proliferation of undifferentiated osteoblasts derived from alveolar bone. It was also confirmed that carrier 2 is also useful for the proliferation of undifferentiated osteoblasts derived from alveolar bone.

Example 12: Osteoblast differentiation on carriers
Differentiation In the same manner as in Example 11 ^{, HAOB3 was seeded on the carrier 5} at 1 × 105 cells / carrier to prepare a composite of HAOB and the carrier. Regarding the differentiation of HAOB into osteoblasts in the obtained complex, the activity of alkaline phosphatase was evaluated by staining as an index of osteoblast differentiation. Specifically, the obtained complex was subjected to MF medium (manufactured by Toyobo Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone at 37 ° C. in the presence of _{5% CO 2.} The cells were cultured, fixed with 10 wt% formaldehyde for 30 minutes, and stained with an alkaline phosphatase stain for 5 minutes.

Further, the experiment was carried out in the same manner except that the carrier 2 (Example 10-2) was used instead of the carrier 5.
Results The results showing changes in alkaline phosphatase activity are shown in FIG. As is clear from FIG. 22, it was confirmed that the carrier 5 became alkaline phosphatase positive from the 3rd day of the culture and increased on the 7th, 14th, and 21st days of the culture. On the other hand, in carrier 2, alkaline phosphatase staining became positive after 7 days of culture. Therefore, it was confirmed that undifferentiated osteoblasts derived from alveolar bone can be differentiated into osteoblasts in any case. Since the differentiation rate of the carrier 5 was faster than that of the carrier 2, it was confirmed that the carrier 5 can more efficiently differentiate undifferentiated osteoblasts derived from alveolar bone into osteoblasts.

Example 13 Analogously to calcification derived Example 11 in carrier seeded HAOB the carrier 2 and 5 at 1 × 10 ⁵ cells / carrier, to prepare HAOB a carrier of the composite, respectively. Alizarin red staining was performed to assess the calcification production of HAOB in the resulting complex. Specifically, the obtained complex was subjected to MF medium (manufactured by Toyobo Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone at 37 ° C. in the presence of _{5% CO 2.} The cells were cultured, fixed with 10 wt% formaldehyde over time for 30 minutes, and stained with 2% Alizarin red S (manufactured by Wako Pure Chemical Industries, Ltd.) for 5 minutes.

Results The change over time in calcium deposition by alizarin red staining is shown in FIG. Deposition of calcification was observed on both carriers 2 and 4. In particular, as is clear from FIG. 23, calcification was observed on the carrier 2 after the 14th day of culturing, but calcification was observed earlier on the carrier 5. From this, it was confirmed that the carrier 5 can efficiently induce calcification by forming a complex with undifferentiated osteoblasts derived from alveolar bone.

From this, it was confirmed that the complex of the carrier 5 and the undifferentiated osteoblasts derived from the alveolar bone is a complex that can efficiently improve the bone defect by transplanting to the bone defect portion.

Example 14: Gene analysis After culturing Example 13, gene expression in the complex was quantified by real-time PCR. Specifically, after culturing for each period according to Example 13 described above, total RNA was extracted from the complex using Isogen according to the method recommended by the manufacturer. CDNA was synthesized from 1 μg of total RNA using reverse transcriptase M-MLV. Then, the mRNA expression levels of the hard tissue formation-related genes, osteolix, type I collagen, Runt-related transcription factor 2 (Runx2), osteocalcin, and bone sialoprotein, were quantified by the real-time PCR method. In addition, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal standard to correct the quantitative analysis. The reaction conditions were 95 ° C., 3 minutes after the reaction, and 45 cycles were performed with 95 ° C., 15 seconds, 55 ° C., 30 seconds, 72 ° C., and 30 seconds as one cycle. The change in each mRNA expression level was calculated by correcting the GAPDH mRNA expression level of the sample by the ΔΔCT method recommended by the manufacturer.

Results The results showing changes in the expression of calcification-related genes in the complex are shown in FIG. As is clear from FIG. 24, no significant difference was observed in the expression of each gene group between carriers 2 and 5 until the 14th day of culture. On the other hand, after the 21st day of culture, it was observed that the carrier 5 was predominantly highly expressed in hard tissue-related genes such as osteolics, osteocalcin, and bone sialoprotein, which induce calcification.

From this, it was confirmed that the complex of the carrier 5 and the undifferentiated osteoblasts derived from the alveolar bone is a complex that can efficiently improve the bone defect by transplanting to the bone defect portion.

From these facts, the carrier of the present invention efficiently proliferates undifferentiated osteoblasts derived from alveolar bone, differentiates into osteoblasts, and calcifies, while having sufficient performance to withstand cell culture operations and the like. It was confirmed that it could be carried out. This enables efficient regeneration of bone tissue, which could not be achieved by conventional techniques, such as regeneration of severe periodontal disease accompanied by a large bone defect.

Example 15: Preparation of carrier
Example 15-1
A cotton-like molded body (low density layer) and a coating layer were formed in the same manner as in Example 10-1. The molded product thus obtained was compressed to a bulk density of about 0.0032 g / cm ³ (thickness of about 2 mm) and heated at 45 ° C. or higher to perform a molding treatment to obtain a carrier. This was designated as carrier 6.

The fibers constituting the carrier 6 have a structure in which hydrophilic polymer fibers are formed inside the hydrophobic polymer fibers, and the hydrophilic polymer fibers have a three-dimensional network structure inside the composite fiber having the structure. Was forming. In the carrier 6, the fibers of the hydrophilic polymers are bonded to each other in this way to form a twisted structure in the composite fibers, and the contact between the composite fibers is controlled. Further, the carrier 6 had a coating layer similar to that in FIG.

Example 15-2
A cotton-like molded body (low density layer) was formed in the same manner as in Example 15-1 except that the spinning time was twice that of Example 15-1, and then a coating layer was formed in the same manner. The molded product thus obtained was adjusted to have a bulk density of about 0.0048 g / cm ³ (thickness of about 2 mm) and subjected to molding treatment to obtain a carrier. This was used as carrier 7. The carrier 7 also formed the three-dimensional network structure recognized in the carrier 6. In addition, the carrier 7 also had a coating layer similar to that in FIG.

Example 15-3
A cotton-like molded body (low density layer) was formed in the same manner as in Example 15-1 except that the spinning time was three times that of Example 15-1, and then a coating layer was formed in the same manner. The molded product thus obtained was adjusted to have a bulk density of about 0.0064 g / cm ³ (thickness of about 2 mm) and subjected to molding treatment to obtain a carrier. This was designated as carrier 8. The carrier 8 also formed the three-dimensional network structure recognized in the carrier 6. Further, the carrier 8 also had a coating layer similar to that in FIG.

Example 15-4
The carrier 7'obtained in the same manner as in Example 15-2 except that the coating layer is formed on only one side so that the coating layer is on the outside (one coating layer is the upper surface and the other coating layer is the lower surface) 2 After stacking them one above the other, they were molded in the same manner as described above ^{to obtain a carrier having a bulk density of 0.0096 g / cm 3} and a thickness of about 2 mm. This was designated as carrier 9. The carrier 9 also formed the three-dimensional network structure recognized in the carrier 6. The carrier 9 also had a coating layer similar to that in FIG.

Example 15-5
The carriers 8'obtained in Example 15-3 were laminated one above the other in the same manner as described above so that the coating layer was on the outside, except that the coating layer was formed on only one side, and then molded in the same manner as described above. A carrier having a bulk density of 0.0192 g / cm ³ and a thickness of 2 mm was obtained. This was designated as carrier 10. The carrier 10 also formed the three-dimensional network structure recognized in the carrier 6. In addition, the carrier 10 also had a coating layer similar to that in FIG.

Example 16: Cell culture on a carrier
Cell culture The carriers 6 to 10 and the carrier 2 are ^{cut into 1 cm 2} × 1 cm ² (thickness is each of the above values), and are immersed in 70 wt% ethanol for sterilization in the same manner as in Example 11. , The cells were allowed to stand in each well of a 24-well plate, immediately washed with PBS (-), and replaced with MF medium. Then, HAOB3 was seeded with 1 × 10 ³ cells / carrier to prepare each complex, and _{cultured in a CO 2} incubator at 37 ° C. for 1, 4 and 7 days. Next, the absorbance was measured over time by the WST8 method (Cell Counting Kit-8, manufactured by Dojin Chemical Laboratory) in the same manner as in Example 11, and cell proliferation was examined.

Results The results are shown in FIG. 25 (the vertical axis shows the absorbance). As is clear from FIG. 25, cell proliferation was observed on all carriers. Compared with the carrier 2, the carriers 6 to 10 suppressed cell proliferation at the initial stage of culturing for 7 days.

Example 17: Osteoblast differentiation and calcification on the carrier
Cut differentiation the carrier 6 to 10 and a carrier 2 in the longitudinal 1 cm ² × horizontal 1 cm ^2, respectively, were seeded HAOB3 with MF medium at 1 × 10 ⁴ cells / carrier in the same manner as in Example 16, HAOB a carrier Each of the above composites was prepared. Regarding the differentiation of HAOB into osteoblasts in the obtained complex, the activity of alkaline phosphatase was evaluated by staining as an index of osteoblast differentiation. Specifically, the obtained complex was subjected to MF medium (manufactured by Toyobo Co., Ltd.) containing 10 mmol / l β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nmol / l dexamethasone at 37 ° C. in the presence of _{5% CO 2.} Incubate for 1, 2 or 3 weeks, fix with 10 wt% formaldehyde over time for 30 minutes, and alkaline phosphatase stain (1M tris-HCl containing _{NN dimethl formamid, Naphthol AS-MX phosphate, Fast blue BB salt, MgCl 2).} Stained for 5 minutes.

Calcification ^{HAOB3 was seeded with 1 × 10 4} cells / carrier using MF medium in the same procedure as in Example 17 to prepare a complex of HAOB and a carrier, respectively. Alizarin red staining was performed to assess the calcification production of HAOB in the resulting complex. Specifically, the obtained complex was cultured for 1, 2 to 3 weeks in the same manner as in Example 13, fixed with 10 wt% formaldehyde for 30 minutes over time, and stained with 2% Alizarin red S. did. The test was conducted in the same manner as described above except that each carrier on which HAOB3 was not seeded was used as a control.

Results The results are shown in FIG.
As is clear from FIG. 26, alkaline phosphatase was positive and differentiation was observed in all carriers. Among these, carriers 6 to 10 had a faster differentiation rate than carrier 2. From this, it was confirmed that the carriers 6 to 10 can differentiate the undifferentiated osteoblasts derived from alveolar bone into osteoblasts more efficiently than the carriers 2. In particular, it was confirmed that undifferentiated osteoblasts derived from alveolar bone can be more efficiently differentiated into osteoblasts on carriers 8 to 10, and further on carriers 9 and 10.

Further, as is clear from FIG. 26, calcification was observed on all the carriers. In addition, among these, desirable calcification was observed earlier in carriers 6 to 10 as compared with carrier 2. When the calcification-induced carrier was stained without seeding the cells, no significant staining was observed as compared with the carrier in which the cells were seeded (each photograph in the second row from the bottom of FIG. 26). From this, it was confirmed that the calcification observed in the carrier in which the cells were seeded was derived from cells (that is, derived from the complex).

From this, it was confirmed that carriers 6 to 10 can induce calcification more efficiently by forming a complex with undifferentiated osteoblasts derived from alveolar bone. In particular, it was confirmed that calcification can be induced more efficiently on carriers 8 to 10, and further on carriers 9 and 10.

This suggests that such a complex of carrier and undifferentiated osteoblasts derived from alveolar bone can efficiently improve bone defect by transplanting to the bone defect portion.

Example 18: Bone regeneration in vivo
The bone regeneration carrier 10 ^{was cut into 1 cm 2} × 1 cm ² in length and 1 × ⁴ cells of HAOB3 were seeded in the same manner as in Example 17 to prepare a complex. Then, the cells were cultured in a bone differentiation-inducing medium (MF medium containing vitamin C, dexamethasone, β-glycerophosphate, and BMP2) for 1 week. After culturing, they were subcutaneously transplanted into immunodeficiency mice (SCID mice). Tissue pieces (tissue containing the transplanted carrier) excised 4 weeks after transplantation were stained with hematoxylin and eosin (HE staining), and their morphology was observed.
Specifically, the tissue piece was fixed with 10 wt% formaldehyde for 24 hours and degreased with 80 wt% methanol and chloroform. Then, it was decalcified with 10 wt% formic acid for 24 hours, washed with water, dehydrated with ethanol according to a conventional method, replaced with remozole, and permeated with paraffin. A 5 μm slice of the sample obtained by embedding was prepared and stained with HE. HE staining was performed by deparaffinizing with remozole and ethanol, then staining the cell nucleus with hematoxylen and eosin, dehydrating with ethanol, and transparently encapsulating with remozole for tissue observation.
As a control, bone regeneration was observed in the same manner as described above except that the carrier 10 not seeded with HAOB3 was used.

Results Figure 27 shows the results. As is clear from the figure, when the carrier 10 seeded with HAOB3 is used (left photo of FIG. 27) as compared with the carrier 10 not seeded with HAOB3 (right photo of FIG. 27), the blood vessels to the carrier are used. Invasion and formation of bone-like structures with osteoblast-like cells around the carrier (arrows in the photograph) were observed. From this, it was confirmed that the complex in which bone formation was observed is useful for bone regeneration in vivo.
From the results of these examples, it was found that the carrier enables long-term culture of undifferentiated osteoblasts.
In addition, compared with the case of using the carrier 2, when the carriers 5 to 10 were used, the proliferation of undifferentiated osteoblasts was suppressed when the complex was cultured for 7 days, that is, at the initial stage of the culture. .. In addition, as compared with the case of using the carrier 2, when the carriers 5 to 10 were used, the induction of differentiation of undifferentiated osteoblasts into bone cells and the induction of calcification were promoted in the complex. From this, it was confirmed that carriers 5 to 10 can more efficiently promote the induction of differentiation of undifferentiated osteoblasts into osteoblasts and the induction of calcification than the carrier 2. Among these, carriers 8 to 10, and carriers 9 and 10, especially carrier 10, were able to more efficiently promote the induction of differentiation and calcification of undifferentiated osteoblasts derived from alveolar bone into osteoblasts. From these facts, it was found that the carriers 5 to 10, particularly carriers 8 to 10, further carriers 9 and 10, and particularly carriers 10, are more useful for bone regeneration from undifferentiated osteoblasts derived from alveolar bone. It was.

According to the present invention, undifferentiated osteoblasts derived from alveolar bone can be efficiently proliferated and / or differentiated on a carrier. Further, according to the present invention, bone tissue derived from undifferentiated osteoblasts derived from alveolar bone can be easily and efficiently produced. According to the present invention, bone tissue can be regenerated to a practical extent.

1: Cotton-like low-density layer 1': Cotton-like low-density layer 1'containing arbitrary components: Cotton-like low-density layer with different polymer composition 2: Coating layer (shape stabilizing layer)
3: High-density layers 3': High-density layers 4 and 4'containing arbitrary components, or having different polymer compositions, or having both characteristics: Carriers having different internal structures and compositions.

Claims (9)

Complex of undifferentiated osteoblasts derived from alveolar bone and carrier:
Here, the carrier is a fiber molded product having a three-dimensional network structure.
The bulk density of the fiber molded product is 0.0001 to 0.25 g / cm ³ .
The fibers constituting the fiber molded product include composite fibers containing a biocompatible hydrophobic polymer and a biocompatible hydrophilic polymer.
The composite fiber has a structure in which the fiber of the hydrophilic polymer is formed inside the fiber of the hydrophobic polymer.
The fibers of the hydrophilic polymer form a three-dimensional network structure inside the composite fiber, and the fibers of the hydrophilic polymer are bonded to each other to form a twisted structure in the composite fiber to bring the composite fibers into contact with each other. Controlling
(Here, the complex is β-TCP (β-tricalcium phosphate), α-TCP (α-tricalcium phosphate), HA (hydroxyapatite), DCPD (second calcium phosphate), OCP (octacalcium). phosphate), 4CP (tetra calcium phosphate), alumina, zirconia, calcium aluminate _{(CaO-Al 2 O 3)} , aluminosilicate _{(Na 2 O-Al 2 O} 3 -SiO 2), bioactive glass, quartz And those containing at least one bone filling material selected from the group consisting of calcium carbonate) . Further, the composite excludes one containing at least one water-soluble polymer selected from the group consisting of hydroxypropyl cellulose, polyethylene glycol, carboxymethyl cellulose, polyvinyl alcohol, hydroxypropyl methyl cellulose and carboxyvinyl polymer. , The composite according to claim 1. The composite according to claim 1 or 2, wherein the fiber molded product has a coating layer at least in part. The composite according to claim 3, wherein the coating layer has a thickness of 1000 μm or less. The composite according to claim 3 or 4 , wherein the coating layer includes a plurality of through holes having an average pore diameter of 10 μm or more. The composite according to claim 5 , wherein the area of the opening formed by the plurality of through holes is 50% or more of the surface area of the coating layer. The composite according to any one of claims 1 to 6 , wherein the thickness of the carrier is 0.05 to 500 mm. The complex according to any one of claims 1 to 7 , wherein at least a part of the undifferentiated osteoblasts derived from alveolar bone is in a state of being differentiated into osteoblasts derived from alveolar bone. The complex according to any one of claims 1 to 7 , wherein at least a part of the undifferentiated osteoblasts derived from the alveolar bone is in a calcified state.

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