Bone filler and prosthetic material

Abstract

To provide a bone filler enabling low invasiveness and promotion of hard tissue growth, and a prosthetic material including the bone filler.SOLUTION: A bone filler used at the time of implantation of an implant body, the bone filler comprising dedifferentiated fat cells and a carrier. Also, a prosthetic material comprising an implant body and dedifferentiated fat cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bone substitute and a prosthetic material. [Background technology]

[0002] When teeth are lost due to periodontal disease, caries, fractures, etc., implant treatment may be performed to improve aesthetic problems, chewing disorders, occlusion disorders, etc. In implant treatment, the implant body is embedded in the jawbone and osseointegration is achieved, allowing the implant to function physiologically as a tooth. However, at the time of implantation, there is often insufficient bone volume to achieve osseointegration.

[0003] Regenerative medicine, which aims to supplement and restore lost tissue, is attracting attention for its potential usefulness in the field of dentistry. Tissue regeneration is thought to require cells (e.g., stem cells), scaffolds, and growth factors, as well as the environment at the transplant site and the time required for regeneration. Many details of the actual mechanism of tissue regeneration remain unclear. In dental implant treatment, when bone mass is insufficient, autologous bone grafts or bone substitute materials (β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), etc.) may be used. In some cases, these treatments also involve the use of growth-promoting factors such as BMP-2 and polycrystalline plasma (PRP).

[0004] Dedifferentiated fat cells (DFAT) are cells induced from mature adipocytes and have the same differentiation potential as stem cells. DFAT can be obtained by isolating mature adipocytes from adipose tissue and subjecting the mature adipocytes to ceiling culture (Patent Documents 1 and 2). DFAT has been reported to be used as a material for periodontal tissue regeneration (Patent Document 3), among other applications. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5055611 [Patent Document 2] Patent No. 5055613 [Patent Document 3] Patent No. 6304715 Summary of the Invention [Problem to be solved by the invention]

[0006] When autologous bone is used as a bone substitute to compensate for bone deficiency during implant placement, it must be harvested from another site, which is highly invasive and limits the amount that can be harvested. When artificial bone primarily composed of β-TCP or HA is used as a bone substitute, the rate of resorption of the artificial bone is slow, and some of the artificial bone may remain. In this case, there is a risk that the remaining artificial bone may become a source of bacterial infection. Therefore, the development of a new bone substitute is required.

[0007] Patent Document 2 describes that packing a periodontal tissue regeneration material containing DFAT into the defect site of a periodontal tissue defect model enables regeneration of cement chambers and regeneration of fibers having periodontal ligament functions. However, Patent Document 2 does not describe that the material can be used as a bone filler when implanting an implant body.

[0008] Therefore, an object of the present invention is to provide a bone substitute material that is less invasive and can promote the growth of hard tissue, and a prosthetic material containing the bone substitute material. [Means for solving the problem]

[0009] The present invention includes the following aspects. [1] A bone filler containing dedifferentiated fat cells and a carrier, used when placing an implant. [2] The bone filler according to [1], wherein the implant body is a dental implant body. [3] A bone filler according to [1] or [2], wherein the carrier is fibrous. [4] A bone filler according to any one of [1] to [3], wherein the carrier comprises at least one selected from the group consisting of β-tricalcium phosphate and lactic acid-glycolic acid copolymer. [5] A prosthetic material comprising an implant body and dedifferentiated fat cells. [6] The prosthetic material according to [5], which is a dental material. [7] The prosthetic material according to [5] or [6], wherein the dedifferentiated fat cells are retained on at least a portion of the surface of the implant body. [8] The prosthetic material according to any one of [5] to [7], further comprising a carrier capable of retaining the dedifferentiated fat cells. [9] The prosthetic material according to [8], wherein the carrier is fibrous.

[10] The prosthetic material according to [8] or [9], wherein the carrier comprises at least one selected from the group consisting of β-tricalcium phosphate and lactic acid-glycolic acid copolymer. [Effects of the Invention]

[0010] According to the present invention, there are provided a bone substitute material that is less invasive and capable of promoting the growth of hard tissue, and a prosthetic material containing the bone substitute material. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing an example of implant treatment. [Figure 2] FIG. 1 is a schematic diagram showing a method for preparing DFAT from adipose tissue in Experimental Example 1. [Figure 3] 1 shows the implant body used in Experimental Example 2. [Figure 4] 1 shows the cotton-like artificial bone filler ReBOSSIS (registered trademark)-J (ORTHOREBIRTH) used in Experimental Example 2. [Figure 5] 3 shows photographs of each step in the preparation of an implant body in Experimental Example 2. [Figure 6] 10 shows photographs of each step in the preparation of a defect model of the control group in Experimental Example 3. [Figure 7]10 shows photographs of each process of producing a defect model of the experimental group in Experimental Example 3. [Figure 8] 1 is a schematic diagram showing a defect model of an experimental group prepared in Experimental Example 3, showing the state in which an implant body of the experimental group is embedded in a defect site. [Figure 9] 1 shows a CT image of the implant body insertion site and its analysis image in Experimental Example 4. 0w: 0 week after implant body insertion; 4w: 4 weeks after implant body insertion. [Figure 10] 1 shows CT images of the implant body insertion site of the experimental group and corresponding analysis images in Experimental Example 4. 0w: 0 week after implant insertion; 2w: 2 weeks after implant insertion; 4w: 4 weeks after implant insertion. [Figure 11] The figure shows the results of quantifying the amount of new hard tissue based on the number of pixels in the area determined to be new hard tissue in the analysis image of the CT image in Experimental Example 4. 2w: 2 weeks after implant placement; 4w: 4 weeks after implant placement. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Bone replacement material] A first aspect of the present disclosure is a bone substitute material used when embedding an implant body. In one embodiment, the bone substitute material comprises dedifferentiated adipocytes and a carrier.

[0013] Figure 1 is a schematic diagram showing an example of dental implant treatment. Figure 1(1) shows the state of a healthy tooth. Figure 1(2) shows the state of a tooth lost due to periodontal disease, tooth decay, or other causes. When performing implant treatment, a hole is first drilled into the jawbone at the site of tooth loss, and the implant body is then inserted (Figure 1(3)). This state is maintained for several months to achieve osseointegration, after which a superstructure is attached (Figure 1(4)). Osseointegration refers to a state in which the implant body, which is a foreign body, is firmly fixed (osseointegrated) within the jawbone.

[0014] The bone graft material according to the first aspect is used when implanting an implant body. "Implanting" refers to drilling a hole in the bone at the site where implant treatment will be performed and embedding the implant body in the hole. The bone graft material is, for example, embedded together with the implant body when the implant body is embedded. The bone graft material is, for example, placed around the implant body at the implantation site of the implant body.

[0015] <Dedifferentiated adipocytes (DFAT)> DFAT is a dedifferentiated cell induced from mature adipocytes, and is a cell with multipotency and proliferation ability. DFAT is a fibroblast-like cell without lipid droplets, obtained by ceiling culture of mature adipocytes, etc. Animals from which DFAT is derived may be any animal with adipose tissue, including humans and non-human mammals. Non-human mammals include, but are not limited to, non-human primates (monkeys, chimpanzees, gorillas, etc.), rodents (mice, rats, guinea pigs, etc.), cats, dogs, rabbits, cows, horses, goats, sheep, pigs, camels, etc.

[0016] DFAT is made from mature adipocytes, which account for approximately 30% of adipose tissue. 8 Approximately 1000 DFAT cells can be obtained. Therefore, it can be said that the collection of adipose tissue, the raw material for DFAR, is minimally invasive. When isolating mature adipocytes from adipose tissue, other cells are less likely to become contaminated, allowing highly pure DFAT cells to be obtained from the early stages of culture. DFAT has a high proliferation potential, and its proliferation potential and pluripotency are not easily affected by the age or underlying diseases of the donor from whom it is collected.

[0017] DFAT may be autologous cells or allogeneic cells. Autologous DFAT is DFAT derived from mature adipocytes collected from an individual who is the subject of implant therapy. Allogeneic cells are DFAT derived from mature adipocytes collected from a donor other than the individual who is the subject of implant therapy. The donor of allogeneic cells may be of any species as long as it is the same species as the individual who is the subject of treatment, and there are no restrictions on age or sex.

[0018] Autologous DFAT cells are preferred because they reduce the risk of immune reactions. DFAT has high proliferation potential, making it possible to prepare large amounts of autologous DFAT cells from a small amount of mature adipocytes. Therefore, sufficient amounts of DFAT for implant treatment can be prepared with minimal invasiveness.

[0019] When using allogeneic DFAT cells, DFAT may be induced using mature adipocytes collected from adipose tissue discarded during surgery. For example, a banking system for allogeneic DFAT cells may be established, and DFAT suitable for the individual to be treated with implants may be appropriately selected from the DFAT stored in the banking system.

[0020] DFAT can be produced using known methods. Examples of methods for producing DFAT include those described in Japanese Patent Nos. 5055611 and 5055613. Examples of methods for producing DFAT include the method shown in Figure 1. First, adipose tissue is separated by collagenase treatment or the like to isolate mature adipocytes. Next, the mature adipocytes are subjected to ceiling culture using a tissue culture flask or the like. After performing ceiling culture for approximately 7 to 14 days, a large number of fibroblast-like adipocytes without lipid droplets are observed. These fibroblast-like cells are DFAT.

[0021] Culture media for animal cells can be used as media for producing DFAT from mature adipocytes. Examples of basal media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Media (MEM), Knockout-DMEM (KO-DMEM), Glasgow Essential Medium (G-MEM), Basal Eagle's Medium (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12 (Advanced DMEM / F12), Iscove's Modified Dulbecco's Medium, Ham's F-10, Ham's F-12, 199 Medium, RPMI1640 Medium, and CSTI303-MSC (Cell Science Institute). Serum (e.g., fetal bovine serum (FBS)), serum substitutes, etc. may be added to these basal media. Examples of serum substitutes include one or more selected from albumin, transferrin, KnockOut Serum Replacement (KSR) (a serum substitute for ES cell culture) (Invitrogen), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The medium may be a basal medium supplemented with one or more selected from lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, inhibitors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like. Specific examples of medium include DMEM medium supplemented with FBS (e.g., 20% v / v). The medium may further contain antibiotics (e.g., streptomycin, penicillin, etc.).

[0022] The culture conditions can be those commonly used for culturing animal cells. Examples of temperature conditions include 20°C or higher and 40°C or lower (preferably 37°C). Examples of carbon dioxide concentrations include 5 v / v% CO2. Culture can be carried out, for example, in a culture device under an atmosphere of 5 v / v% CO2. The seeding density of human mature adipocytes is 100 cells / mm 2More than 400cells / mm 2 The culture period can be from 3 to 20 days, preferably from 7 to 14 days. If possible, it is preferable to change the medium every 5 to 10 days.

[0023] DFAT is CD31-negative, CD45-negative, and HLA-DR-negative. CD31, CD45, and HLA-DR are cell surface antigens that can be detected, for example, by flow cytometry, cell staining, etc. Therefore, the purity of DFAT may be confirmed by detecting these cell surface antigens. For example, in flow cytometry using a fluorescently labeled antibody, if a cell is detected that emits stronger fluorescence compared to a negative control (isotype control), the cell is determined to be "positive" for the cell surface antigen. Examples of timing for detecting the cell surface antigen include immediately after DFAT is prepared from mature adipocytes, during subculture of DFAT, and before preparation of a bone substitute.

[0024] DFAT can be used after being subcultured at least once, and the amount of DFAT required for preparing a bone substitute can be obtained by expanding it through subculture.

[0025] Subculture of DFAT can be carried out by known methods. Examples of media used in subculture include those similar to those described above. For subculture, adherent culture (adherent culture) is preferably carried out. Adherent culture can be carried out, for example, using a culture vessel with an adhesive layer provided on the bottom surface. The adhesive layer can be formed on the bottom surface of the culture vessel using an extracellular matrix such as laminin, fibronectin, type I collagen, or gelatin. Culture conditions such as culture temperature and carbon dioxide concentration can be the same as those described above. The interval between subcultures can be 3 to 20 days, preferably 4 to 14 days.

[0026] <Carrier> The carrier is preferably bioabsorbable and capable of receiving DFAT. Examples of the carrier include materials used as artificial bone materials. Examples of the carrier include ceramics such as hydroxyapatite (HA), calcium phosphate (β-TCP, etc.), calcium carbonate, coral, and calcium sulfate; and biodegradable resins such as poly-L-lactic acid (PLLA) and lactic acid-glycolic acid copolymer (PLGA).

[0027] The carrier preferably has a form that can serve as a scaffold for DFAT and allows DFAT to come into contact with the implant body. Examples of the shape of the carrier include, but are not limited to, fibrous (including cotton-like), particulate, sponge-like, and sheet-like shapes. The carrier is preferably fibrous because it is easily fixed to the implant body. When the carrier is fibrous, the fiber diameter is preferably, for example, 1.000 mm or less, more preferably 0.500 mm or less, and more preferably 0.100 mm or less. The lower limit of the fiber diameter is not particularly limited, but may be 0.001 mm or more from the viewpoint of ease of handling. Having a fiber diameter of the carrier that is equal to or less than the above-mentioned preferred upper limit can increase the contact area between DFAT and the implant body. The carrier is preferably a cotton-like carrier as shown in FIG. 4.

[0028] The carrier preferably comprises at least one selected from the group consisting of ceramics and biodegradable resins, more preferably ceramics and biodegradable resins. The carrier preferably comprises at least one selected from the group consisting of calcium phosphate, PLLA, and PLGA, more preferably calcium phosphate and at least one biodegradable resin selected from the group consisting of PLLA and PLGA. The carrier preferably comprises at least one selected from the group consisting of β-TCP and PLGA, more preferably β-TCP and PLGA.

[0029] The carrier can be manufactured by a known method. For example, a spinning solution prepared by mixing a biodegradable resin solution with ceramic particles can be formed into fibers by a known spinning technique (electrospinning, wet spinning, etc.), thereby producing a fibrous carrier. The fibrous carrier can be manufactured by the methods described in, for example, International Publication No. 2015 / 005205, JP 2015-178043 A, JP 2017-857 A, JP 2022-110081 A, etc.

[0030] The carrier may be a commercially available artificial bone material. Examples of commercially available fibrous carriers include the ReBOSSIS (registered trademark) series (ORTHOREBIRTH Co., Ltd.), which is a cotton-like artificial bone filler.

[0031] <Other ingredients> The bone substitute may contain other components in addition to DFAT and a carrier. Examples of other components include a culture medium. The culture medium is not particularly limited as long as it allows DFAT to survive and grow. Examples of the culture medium include the same culture medium as described above.

[0032] <Method for preparing bone substitute material> DFAT used in preparing a bone substitute can be collected from a culture vessel by known methods. For example, DFAT adhering to the culture vessel can be released with an enzyme such as trypsin, and then collected by filtration or centrifugation.

[0033] After collection, DFAT may be washed. Washing of DFAT can be carried out by known methods. For example, the above-mentioned medium, buffer solution, etc. can be used to wash DFAT. DFAT can be washed by suspending it in a washing solution (medium, buffer, etc.) and collecting DFAT from the suspension by centrifugation, etc.

[0034] The bone filler can be prepared by mixing DFAT and a carrier in a culture medium. The same culture medium as described above can be used. The bone filler can be prepared, for example, by suspending DFAT in a culture medium to prepare a DFAT suspension, adding a carrier to the DFAT suspension, and mixing. Alternatively, the bone filler can be prepared, for example, by immersing a carrier in a culture medium, seeding DFAT therein, and mixing. The seeding density of DFAT per 1 g of carrier is, for example, 1.0 × 10 3 pieces / g~1.0×10 8 The number of particles / g is 1.0 x 10 4 pieces / mL~1.0×10 7 The amount of medium used can be, for example, such that the entire carrier comes into contact with the medium.

[0035] The bone filler may be used immediately after mixing DFAT and a carrier in a culture medium. Alternatively, the bone filler may be cultured for an arbitrary period of time after mixing DFAT and a carrier in a culture medium to allow DFAT to adhere to the carrier. Examples of the culture period include 1 minute to 3 hours, 2 minutes to 2 hours, 3 minutes to 1 hour, and 5 minutes to 0.5 hours. The upper and lower limits can be combined arbitrarily. Alternatively, the bone filler may be stored for any period of time in the form of a mixture of DFAT, a carrier, and a culture medium. Examples of storage temperatures include 4 to 10°C, and preferably 4 to 6°C. Alternatively, the bone filler may be a dried mixture of DFAT, a carrier, and a culture medium. Any drying method may be used as long as it does not kill DFAT, and examples of such drying include freeze-drying.

[0036] <How to use bone substitute material> The bone filler is used together with the implant body when the implant body is embedded. The implant body is preferably a dental implant body. Any known implant body can be used without any particular limitation. Examples of the material for the implant body include pure titanium and titanium alloys. The implant body may be a one-piece type or a two-piece type.

[0037] The bone filler is preferably embedded together with the implant body while being held on the surface of the implant body, thereby allowing the DFAT contained in the bone filler to adhere to the surface of the implant body.

[0038] The amount of bone filler used is preferably such that the entire surface of the implant body is covered with the bone filler. Examples of the amount of bone filler used per implant body include 0.01 g or more and 5 g or less, 0.05 g or more and 3 g or less, 0.1 g or more and 2 g or less, 0.2 g or more and 1 g or less, and 0.2 g or more and 0.5 g or less. The above upper and lower limits can be combined arbitrarily.

[0039] The implant body may be cultured in the presence of a bone prosthetic material before implantation. The culture is preferably carried out with the bone prosthetic material retained on the surface of the implant body. By culturing the implant body in the presence of a bone prosthetic material, DFAT can be attached to the surface of the implant body. When the carrier is fibrous, the carrier (bone prosthetic material) to which DFAT has been attached may be wrapped around the implant body. By wrapping the bone prosthetic material around the implant body, the bone prosthetic material can be fixed to the implant body. This makes it easier for DFAT to be retained on the surface of the implant body.

[0040] The culture medium used for culturing the implant body and bone substitute material may be the same as those described above. The culture conditions may be, for example, those typically used for culturing animal cells. The temperature may be, for example, 20°C or higher and 40°C or lower (preferably 37°C). The carbon dioxide concentration may be, for example, 5 v / v % CO2. The culture may be carried out, for example, in a culture device under an atmosphere of 5 v / v % CO2. The culture may also be carried out under air atmosphere.

[0041] The incubation time for the implant body and the bone filler is preferably long enough to allow DFAT in the bone filler to adhere to the surface of the implant body. Examples of incubation times include 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, and 6 hours or more. The upper limit of the incubation time is not particularly limited, but may be, for example, 12 hours or less, or may be 10 hours or less, or 8 hours or less. Examples of incubation times include 1 to 12 hours, 2 to 10 hours, 3 to 8 hours, and 4 to 8 hours. The upper and lower limits can be combined in any manner.

[0042] The implant body can be implanted by a conventional method, except that it is implanted together with a bone graft. Specifically, a hole is drilled in the bone at the implant body implantation position, and the implant body is implanted together with the bone graft. At this time, it is preferable that the bone graft is retained on at least a portion of the surface of the implant body. When culturing is performed with the bone graft retained on the surface of the implant body, the implant body after culturing can be used for implantation as is.

[0043] After implantation, the implant may be fixed with an absorbable membrane or the like, as appropriate. The absorbable membrane is a membrane that has the property of being decomposed or absorbed in the living body. Examples of absorbable membrane materials include extracellular matrices such as collagen; and biodegradable resins such as PLLA and PGLA. Commercially available absorbable membranes include Koken Tissue Guide (collagen membrane, Koken Co., Ltd.), Biomend (collagen membrane, Hakuho Co., Ltd.), GC Membrane (lactic acid-glycolic acid copolymer membrane, GC Corporation), Vicryl Mesh (polyglactin, J&J), INION GTR (registered trademark) (PLLA, INION), and BIO-GUIDE (registered trademark) (collagen, GEISTLICH-PHARMA).

[0044] After the implant body is placed, in the case of a two-piece implant, the apatment is connected. After that, it is left for one to several months for osseointegration, and then the superstructure is attached (see Figure 1). This completes the implant treatment.

[0045] The bone graft material according to the first aspect is embedded together with the implant body when the implant body is implanted, thereby promoting the regeneration of hard tissue around the implant body. Therefore, the bone graft material according to this aspect can be suitably used when there is insufficient bone mass for the implant body to be implanted. Furthermore, the use of the bone graft material according to this aspect is expected to shorten the time required to achieve osseointegration.

[0046] Traditionally, dental pulp stem cells, bone marrow stem cells, and the like have been considered as cells to be used in regenerative medicine in dentistry. Dental pulp stem cells can be collected from wisdom teeth or primary teeth and are minimally invasive. Dental pulp stem cells have high cell proliferation potential but suffer from the problem of low cell stability. Bone marrow stem cells are collected from the hip bone and are highly invasive. Bone marrow stem cells also suffer from the problems of low cell proliferation potential and low cell stability. On the other hand, DFAT can be prepared from abdominal adipose tissue or the buccal fat pad present in the midface. DFAT has high cell proliferation and high cell stability, allowing it to grow in large quantities from a small amount of adipose tissue. Furthermore, as shown in the examples below, bone substitute materials containing DFAT have the effect of promoting the regeneration of hard tissue around the implant body. For these reasons, bone substitute materials containing DFAT can be said to be suitable bone substitute materials for implant treatment.

[0047] [Prosthetic materials] A second aspect of the present disclosure is a prosthetic material. In one embodiment, the prosthetic material includes an implant body and a DFAT. The prosthetic material is preferably a dental material.

[0048] DFAT can be prepared as described above in [Bone Filler]. The implant body is not particularly limited, and the same implant body as described above in [Bone Filler] can be used. The implant body is preferably a dental implant body.

[0049] In the prosthetic material, DFAT is preferably retained on at least a portion of the surface of the implant body. DFAT is preferably retained on a carrier capable of retaining DFAT, and is retained on the surface of the implant body. Examples of the carrier include those similar to those listed above in [Bone Filler]. The carrier is preferably fibrous. In the case of a fibrous carrier, the DFAT-retaining carrier can be wrapped around the implant body to retain DFAT in the implant body. The carrier preferably contains at least one selected from the group consisting of β-TCP and PLGA, and more preferably contains β-TCP and PLGA.

[0050] The prosthetic material can be prepared by the same method as the method for preparing an implant before insertion explained in the above [Bone Filler] <Method of Using the Bone Filler>.

[0051] The prosthetic material can be used immediately after preparation. Alternatively, the prosthetic material may be stored with DFAT attached to at least a portion of the surface of the implant body. The storage temperature is, for example, 4 to 10°C, preferably 4 to 6°C.

[0052] The prosthetic material according to this aspect contains DFAT, and therefore promotes the regeneration of hard tissue around the implant body when embedded in bone, making it suitable for use as a prosthetic material in implant treatment.

[0053] [Other aspects] In one aspect, the present disclosure provides a method for implanting an implant body, comprising implanting the implant body together with DFAT into a bone of a subject. Preferably, DFAT is held on at least a portion of the surface of the implant body. Preferably, DFAT is held on at least a portion of the surface of the implant body by a carrier. The carrier is preferably a fibrous carrier, and examples thereof include those similar to those described above.

[0054] In one aspect, the present disclosure provides the use of DFAT in the manufacture of a bone substitute material for use in implantation of an implant body. In one aspect, the present disclosure provides use of DFAT and a carrier in the manufacture of a bone substitute material for use in embedding an implant body. The carrier is preferably a fibrous carrier, and examples thereof include those similar to those described above. [Example]

[0055] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0056] [Experimental Example 1] (Preparation of DFAT) Subcutaneous adipose tissue was collected from both inguinal regions of F344 rats (8 weeks old, male) (CLEA Japan) and washed. The subcutaneous adipose tissue was then enzymatically treated with 0.1% collagenase (SIGMA) solution. After removing excess tissue using a 100 μm cell strainer (BD Falcon), the tissue was centrifuged at 135 G for 3 minutes, and the mature adipocyte fraction floating at the top of the centrifuge tube was collected.

[0057] The collected mature adipocytes were washed three times with Dulbecco's Modified Eagle Medium (DMEM medium: SIGMA). 2 The flask was filled with DMEM medium supplemented with 20% Fetal Bovine Serum (FBS: Nichirei Biosciences, Inc.) and 1% Pen Strep (GIBCO), and 5 × 10 4Mature adipocytes were seeded onto the inverted flask. The mature adipocytes were allowed to adhere to the ceiling of the flask and subjected to ceiling culture. Ceiling culture was continued for 12 days to obtain DFAT (see Figure 2).

[0058] The required number of cells was obtained by passage of DFAT using trypsin-EDTA solution according to the usual method. In this preparation of DFAT, the required number of cells was obtained by one passage and culturing for 3 to 4 weeks.

[0059] [Experimental Example 2] (Implant body preparation) The implant body used in the test is shown in Figure 3. Pure titanium type 4 implant bodies were used.

[0060] The carrier used in the test is shown in Figure 4. ReBOSSIS (registered trademark)-J (ORTHOREBIRTH), a cotton-like artificial bone filler, was used as the carrier.

[0061] <Preparation of implants for the experimental group> Figure 5 shows photographs of each step in the preparation of the implant body. DFAT in the culture flask was detached by trypsin treatment and then collected by centrifugation (Figure 5(1)). 6 DFAT was suspended in DMEM medium (0.1 mL) and seeded onto one package of ReBOSSIS J (0.5 g), allowing the DFAT to adhere to the ReBOSSIS J. The ReBOSSIS J with DFAT attached was wrapped around the implant body (Figure 5(2)). Next, the implant body wrapped with ReBOSSIS J was cultured in DMEM medium at 37°C for 6 hours (Figure 5(3)).

[0062] <Preparation of implant body for control group> The implant bodies were prepared in the same manner as in the experimental group, except that ReBOSSIS J was not seeded with DFAT.

[0063] [Experimental Example 3] (Creating a defect model) F344 rats (8 weeks old, male) from which subcutaneous adipose tissue had been harvested in Experimental Example 1 were used as the defect model animals. The control group implant was placed in the right femur, and the experimental group implant was placed in the left femur.

[0064] <Creating a control group defect model> Figure 6 shows photographs of each step in the creation of the defect model. First, the right femur of the rat was exposed (Figure 6(1)). Next, a hole 2 mm in diameter and 3 mm deep was drilled using a micromotor 10 mm from the femoral epiphysis to create a defect (Figure 6(2)). Next, the control implant prepared in Experimental Example 2 was embedded in this defect (Figure 6(3)). Next, the implant was fixed in place using an absorbable membrane (BIOMEND, ZIMMER BIOMET) (Figure 6(4)). Next, the fascia was sutured (Figure 6(5)), and then the skin was sutured (Figure 6(6)).

[0065] <Preparation of the experimental group defect model> Figure 7 shows photographs of each step in the preparation of the defect model. First, the left femur of the rat was exposed (not shown). Next, a hole 2 mm in diameter and 3 mm deep was drilled using a micromotor 10 mm from the femoral epiphysis to create a defect (Figure 7(1)). Next, the implant body of the experimental group prepared in Experimental Example 2 was embedded in this defect (Figure 7(2)). Next, the implant body was fixed using an absorbable membrane (BIOMEND, ZIMMER BIOMET) (Figure 7(3)). Next, the fascia was sutured (Figure 7(4)), and then the skin was sutured (Figure 7(5)).

[0066] Figure 8 is a schematic diagram showing the state in which an implant body of the experimental group was embedded in a defect site in the defect model of the experimental group. An implant body of 1 mm in diameter and 2 mm in height was embedded in a defect site of 2 mm in diameter and 3 mm in depth. ReBOSSIS J with DFAT attached was present around the implant body.

[0067] [Experimental Example 4] (CT analysis of implant placement site) CT scans of the implant site were taken 0, 2, and 4 weeks after implant placement (R_mCT, Rigaku Corporation), and image analysis was performed (i-View, Morita).

[0068] Figure 9 shows CT images of the implant site at 0 weeks (0w) and 4 weeks (4w) after implantation, as well as an analysis image of the CT image at 4 weeks. In the control group, new hard tissue was formed on the upper surface of the implant, but no new hard tissue was observed in the abdominal area of ​​the implant. In contrast, in the experimental group, new hard tissue was observed in the surrounding area, including the abdominal area of ​​the implant.

[0069] Figure 10 shows CT images and analysis images of the implant site in the experimental group. These are CT images taken 0 weeks (0w), 2 weeks (2w), and 4 weeks (4w) after implantation, as well as analysis images of the CT images taken 2 and 4 weeks after implantation. Two weeks after implantation, new hard tissue was observed in areas other than the joint side. These results suggest that new hard tissue may be generated at an early stage after implantation.

[0070] Figure 11 shows the results of quantifying the amount of newly formed hard tissue based on the number of pixels in the area identified as newly formed hard tissue in the analyzed CT images. The amount of newly formed hard tissue was greater in the experimental group than in the control group both two and four weeks after implant placement. No significant differences were observed between two and four weeks after implant placement.

[0071] From the above results, it was confirmed that the use of DFAT during implant placement can promote the growth of new hard tissue around the implant. [Industrial Applicability]

[0072] According to the present invention, there are provided a bone substitute material that is less invasive and capable of promoting the growth of hard tissue, and a prosthetic material containing the bone substitute material.

Claims

1. A bone filler used when placing an implant, comprising dedifferentiated fat cells and a carrier.

2. The bone substitute material according to claim 1 , wherein the implant body is a dental implant body.

3. The bone substitute according to claim 1 or 2, wherein the carrier is fibrous.

4. 3. The bone substitute according to claim 1, wherein the carrier comprises at least one selected from the group consisting of β-tricalcium phosphate and lactic acid-glycolic acid copolymer.

5. A prosthetic material comprising an implant body and dedifferentiated fat cells.

6. The prosthetic material according to claim 5, which is a dental material.

7. The prosthetic material according to claim 5 or 6, wherein the dedifferentiated fat cells are retained on at least a portion of the surface of the implant body.

8. The prosthetic material according to claim 5 , further comprising a carrier capable of retaining the dedifferentiated adipocytes.

9. The prosthetic material of claim 8 , wherein the carrier is fibrous.

10. 10. The prosthetic material according to claim 8, wherein the carrier comprises at least one selected from the group consisting of β-tricalcium phosphate and lactic acid-glycolic acid copolymer.

Images