WO2002040071A1

WO2002040071A1 - Compositions for forming bone or periodontium and injections for forming bone or periodontium

Info

Publication number: WO2002040071A1
Application number: PCT/JP2001/009800
Authority: WO
Inventors: Minoru Ueda; Yasuhiro Okazaki; Ken-Ichiro Hata; Youichi Yamada
Original assignee: Osteogenesis Co., Ltd.
Priority date: 2000-11-14
Filing date: 2001-11-08
Publication date: 2002-05-23
Also published as: JPWO2002040071A1; AU2002212739A1

Abstract

Compositions for forming a bone or a periodontium being excellent in handling properties and safety whereby a defect in a bone or a periodontium can be effectively repaired and regenerated without collecting autologous bone. A gel composition is prepared by mixing PRP (platelet-rich plasma) with ß-TCP having an average grain size of 1 to 5 νm and then stirring the obtained mixture together with thrombin and a calcium chloride solution.

Description

Composition for forming bone or periodontal tissue, and injection for forming bone or periodontal tissue

Technical field

The present invention relates to a composition for forming bone or periodontal tissue that can be used for repairing or regenerating bone or periodontal tissue. Light

Field background technology

Various techniques have been developed to repair or regenerate bone tissue and periodontal tissue damage and defects. As one of them, a method using Platelet-rich Plasma (PRP) has been attempted. PRP is a platelet-rich plasma and is prepared by centrifuging the collected blood under specified conditions (Dean H. Whitman et al .: J Oral Maxillofac Surg, 55, 1294-1299 (1997)) . Conventionally, fibrin glue (Fiblin Glue) has been widely used as a healing promoter after surgery (Renato Saltz et al .: Plastic And Reconstructive Surgery, 88, 1005-1015 (1991)). Studies have been conducted to promote the healing of wounds and to improve the effects of bone grafting, etc., by using it as an alternative to detoxification (Dean H. Whitman et al .: J Oral Maxillofac Surg, 55, 1294 ") 1299 (1997)).

The field of application of PRP is wide, and it is applied to bone reconstruction in fields such as otolaryngology, oral surgery, and maxillofacial surgery (Eduardo Anitua: Int Oral Maxillofac Inplants, 14,529-535 (1999), Dean H. Whitman et al .: J Oral Maxillofac Surg, 55, 1294-1299 (1997)). PRP is rich in growth factors such as Platelet-derived Growth Factor (PDGF), Transforming growth factor β1 (TGF-β1), and Transforming growth factor β2 (TGF-) 32). It is thought that growth factors act in combination to promote bone tissue regeneration, etc. (Marx RE. Et al .: Oral Surg Oral Med Oral Pathol, 85, 638-646 (1998)).

As for PDGF, PDGF is a glycoprotein having a molecular weight of about 30 kD and has been confirmed to have an effect of promoting wound healing. PDGF promotes cell mitosis, improves blood circulation by promoting the formation of capillaries, or promotes differentiation and proliferation of fibroblasts, osteoblasts, etc. by enhancing the effects of other growth factors. Is thought to promote wound healing.

On the other hand, in recent years, implant treatment has been performed for alveolar atrophy, but bone transplantation may be necessary in advanced alveolar atrophy cases. As an example, the treatment of maxillary alveolar ridge atrophy involves maxillary sinus floor elevation with bone grafting. However, since the healing period of maxillary sinus elevation is long, the current treatment period for implant treatment is prolonged. Recently, it has been reported that PRP was used in bone transplantation for jaw bone defects, and that this shortened the healing period (Marx RE. Et al .: Oral Surg Oral Med Oral Pathol, 85, 638-646 (1998)). ) _¾ are shown utility of using PRP even fin plant treatment. Disclosure of the invention

As described above, although clinical research using PRP has been conducted, clinical application has preceded it, and basic research has not reported on bone kinetics. At present, there is an urgent need to develop a method for regenerating bone and periodontal tissues that can be applied to actual clinical practice. Such a regeneration method is required not only to have a high bone or periodontal tissue regeneration effect, but also to have high operability and safety.

Therefore, the present invention aims at providing a composition for forming bone or periodontal tissue used in the method in order to provide a novel method for regenerating bone or periodontal tissue which can be applied to clinical practice. Target.

The present inventors have conducted intensive studies in view of the above problems. First, when a transplant material was constructed by combining PRP and an inorganic bioabsorbable material, and bone regeneration was examined using this material, the bone inducing ability for cells existing in the adaptation site at the application site was amplified. And a high bone regeneration effect was found. It was also suggested that inclusion of a particulate inorganic bioabsorbable material in PRP facilitates appropriate plasticity and shape retention.

On the other hand, when PRP and cells having osteogenic ability were mixed and gelled, and bone regeneration was attempted, a favorable bone regeneration effect was observed. Furthermore, when bone regeneration was attempted using a graft material in which fibrinogen, an inorganic bioabsorbable material, and cells having bone forming ability were mixed, a high bone regeneration effect was observed. In addition, when bone formation was attempted using a graft material obtained by mixing PRP, alginate (alginate), and cells capable of forming bone, a favorable bone regeneration effect was observed.

The present invention is based on the results of the above study, and has the following configuration. That is, a first aspect of the present invention is a composition for forming a bone or periodontal tissue, which comprises PRP and an inorganic bioabsorbable material and has fluidity during use. Further, a second aspect of the present invention is a composition for forming a bone or periodontal tissue having fluidity at the time of use, comprising PRP and cells having an osteogenic ability.

Still another aspect of the present invention is a composition for forming a bone or periodontal tissue having fluidity during use, comprising fibrinogen, an inorganic bioabsorbable material, and cells having bone forming ability. It is. Still another aspect of the present invention is a composition for forming bone or periodontal tissue having fluidity during use, comprising PRP, alginate (alginate), and cells having bone forming ability. It is.

Here, the fluidity of the composition for forming bone or periodontal tissue depends on the injection container at the time of use. It is preferable to have fluidity that can be used by injection.

The composition for forming bone or periodontal tissue of the present invention can be prepared further containing a gelling material. Thrombin and calcium chloride can be used as the gelling material.

The composition for forming bone or periodontal tissue of the present invention can be used after being frozen once and then thawed from the frozen state at the time of use.

On the other hand, the composition for forming bone or periodontal tissue can be sealed in an injection container to form an injection for forming bone or periodontal tissue.

The inorganic bioabsorbable material in the present invention includes one selected from the group consisting of: 3-tricalcium phosphate, α-tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. Alternatively, two or more inorganic bioabsorbable materials can be used. As the inorganic bioabsorbable material, those having an average particle diameter of 0.5 m to 50 / m can be used. Further, the content of the inorganic bioabsorbable material can be, for example, 30% by weight to 75% by weight.

According to the configuration of the present invention as described above, since PRP containing a growth factor rich in promoting proliferation and differentiation of osteogenic cells is contained, the growth site can be used at an application site (transplant site) by these growth factors. Effective regeneration of bone tissue or periodontal tissue can be expected. In addition, since it has fluidity at the time of use, it is versatile because it is not necessary to pre-mold it in accordance with the shape of the bone or periodontal tissue defect, and its handling is easy.

In addition, the use of autologous PRPs allows the application of non-toxic and immune-inactive growth factors, and their safety is high.

In addition, by using the inorganic bioabsorbable material together, it becomes a scaffold for bone cells and the like at the application site, and can further promote regeneration of bone tissue and the like. In addition, inorganic bioabsorbable materials will be replaced by bone tissue in the future. Its safety is high.

^ On the other hand, by including cells having osteogenic ability, direct regeneration of bone tissue by itself can be expected.

As described above, according to the configuration of the present invention, a bone or periodontal tissue defect can be effectively repaired and regenerated without collecting autologous bone, and bone or bone having high operability and safety can be obtained. A composition for periodontal tissue formation is provided. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows a photograph of the results of the PRP group in Example 4 (right side of the maxillary sinus implanted with the PRP gel containing / 3-TCP) two weeks after transplantation. Gray HE (Hematoxylin Eosin) stained histology is shown. 3) It is observed that 3-TCP accumulates and fills the transplanted bone, and a large number of osteoblasts surround it. Osteoblasts are also observed in 13-TCP. Juvenile new bone is observed around some | 8-TCP.

FIG. 2 is a photograph showing the results of the 4 weeks after transplantation of the PRP group (the right side of the maxillary sinus transplanted with the PRP gel containing / 3-TCP) in Example 4; Gray HE (Hematoxylin Eosin) stained histology is shown. I3-TCP has decreased compared to 2 weeks after transplantation. ) It is observed that osteoblasts are encapsulated all around the 3-TCP. In addition, the formation of new bone is observed around 3-TCP. It is observed that many osteoblasts also exist around the new bone. There is a cement line in the new bone.

FIG. 3 is a photograph showing the results of the control group in Example 4 (left side of the maxillary sinus to which only I3-TCP was transplanted) two weeks after transplantation. Hematoxylin eosin) The stained histology is shown. ) 3-TCP is present in the transplanted bone as a square, and osteoblasts are observed around J3-TCP. Also, Slight formation of new bone is observed.

FIG. 4 is a photograph showing the results of the control group in Example 4 (left side of the maxillary sinus transplanted with only | 8-TCP) 4 weeks after transplantation. (Hematoxylin eosin) The stained tissue image is shown. Many / 3 -TCPs are observed in blocks at the implant site, and new bone formation is observed around them. A cement line is found in the trabecular bone of the new bone.

Fig. 5 is a photograph showing the results of the PRP group in Example 4 (the right side of the maxillary sinus transplanted with the PRP gel containing / 3-TCP) 8 weeks after transplantation, and demineralization of the transplanted portion 8 weeks after transplantation. HE (hematoxylin eosin) stained histology is shown. Increased bone mass is seen at and around the implant.

Figure 6 is an enlarged (16x) view of a portion of Figure 5. The formation of new bone (B) is observed so as to be in contact with the i3-TCP-containing PRP gel implant (A).

Fig. 7 is an enlarged (16x) view of a part of Fig. 5, similar to Fig. 6. As in FIG. 6, formation of new bone (B) is observed so as to be in contact with the 3-TCP-containing PRP graft (A). FIG. 8 is a photograph showing the results of the control group in Example 4 (left side of the maxillary sinus transplanted with -TCP alone) at 8 weeks after transplantation. (Matoxylinje) A stained histological image is shown. There is almost no increase in bone mass compared to 4 weeks after transplantation.

Figure 9 is an enlarged (16x) view of part of Figure 8. It is observed that the fibrous connective tissue (c) surrounds the j3-TCP gel implant (a). Bone (b) is outside fibrous connective tissue (c).

FIG. 10 is a graph summarizing the results of Example 4, and shows the ratio (%) of the ossified portion to the entire transplanted portion. The amounts of newly formed bone and the amount of 3-TCP remaining at 2, 4 and 8 weeks after transplantation are shown. In the figure, (+) indicates the results of the PRP group, and (1) indicates the results of the control group. 8 weeks after transplantation It can be seen that the amount of new bone in the test group is significantly higher than that in the control group. It is also observed that the PRP group absorbed 0-TCP better.

FIG. 11 is a photograph showing two weeks (2 W), four weeks (4 W), and eight weeks (8 W) after transplantation of the PCBM transplanted part in Example 8, and demineralization of the transplanted part. HE (hematoxylin eosin) stained histology is shown.

FIG. 12 is a diagram showing photographs at two weeks (2 W), four weeks (4 W), and eight weeks (8 W) after the transplantation of the PRP-cell gel-transplanted part in Example 8. The decalcified HE (hematoxylin eosin) stained histology is shown.

FIG. 13 is a diagram showing photographs of the results at 2 weeks (2 W), 4 weeks (4 W), and .8 weeks (8 W) after the transplantation of the PRP gel transplant portion in Example 8. The decalcified HE (hematoxylin eosin) stained histology is shown.

FIG. 14 is a view showing a photograph of the result of the control group in Example 8. Photographs at 2 weeks (2 W), 4 weeks (4 W), and 8 weeks (8 W) of the defect left untransplanted, with decalcified HE (hematoxylin and eosin) staining The histology is shown. FIG. 15 is a photograph showing a directly observed transplantation site 8 weeks after the transplantation of the transplantation composition (fibrinogen-j3-TCP-cell-containing composition). A white lump is observed at the transplant (arrow).

Fig. 16 shows the HE-stained tissue images at 2 weeks (2W), 4 weeks (4W), and 8 weeks (8W) after transplantation of the transplantation composition (fibrinogen-) 3-TCP-cell-containing composition. FIG. Two weeks after transplantation (2W), lining with osteoblasts is seen (arrow), indicating that bone formation is taking place. Four weeks after transplantation (4W), osteocytes became visible (arrows), indicating that active bone formation was progressing. At 8 weeks (8W) after transplantation, a partial laminar structure is seen (arrow), and bone maturation can be observed.

FIG. 17 is a view showing an HE-stained tissue image of the control group with the transplantation composition (fibrinogen- | 3-TCP-cell-containing composition) at 8 weeks after transplantation. Slightly Bone formation is observed.

FIG. 18 (A) is a diagram showing an H.E.-stained tissue image of the transplanted part 16 weeks after transplantation of (PRP-alginate-one-cell-containing composition). FIG. 18 (B) is a view showing an H.E.-stained tissue image of the transplanted portion 16 weeks after transplantation of the control group (transplanted with alginate). In the group (A) into which the composition for transplantation was transplanted, new bone was observed (arrow), and it was confirmed that bone regeneration was performed. On the other hand, in the control group (B), the transplanted alginate is observed by staining (arrow), but no new bone formation is observed. BEST MODE FOR CARRYING OUT THE INVENTION

PRP refers to platelet-rich plasma, that is, a platelet-rich plasma. In other words, it refers to platelet-enriched plasma. PRP is prepared, for example, by subjecting collected blood to centrifugation according to the method of Whitman et al. (Dean H. Whitman et al .: J Oral Maxillofac Surg, 55, 1294-1299 (1997)). can do. PRP is known to be rich in growth factors such as Platelet-derived Growth Factor (PDGF), Transforming growth factor β1 (TGF-β1), and Transforming growth factor β2 (TGF-β2). darry J. Peterson: Oral surg Oral Med Oral Pathol Oral Radiol Endod, 85, 638-646 (1998)).

Here, an example of a method for preparing PRP is as follows. First, an anticoagulant such as sodium citrate is added to the collected blood and left at room temperature for a predetermined time. Thereafter, centrifugation is performed under conditions (for example, about 5,400 rpm) at which blood cells and buffy coat separate. This separates into two layers (the upper layer is called platelet-poor plasma; the lower layer contains blood cells and buffy coat). After removing the upper layer, centrifugation is further performed under conditions that allow red blood cells to be separated (for example, about 2,400 rpm). The resulting fraction substantially free of red blood cells (Platelet-rich Plasma: PRP) is collected. How to prepare PRP The method is not limited to this method, and can be prepared by a method modified as necessary.

There is no general definition of the amount of platelets contained in PRP, but plasma containing about 5 to about 20 times platelets compared to the collected blood can be used as PRP in the present invention. . In addition, as long as the preparation is possible and there is no unreasonable burden in the preparation, it is preferable to use a platelet having as large a content of platelets as possible.

Preferably, autologous PRP is used. This eliminates the risk of toxicity or immune rejection.

PRP is rich in platelet, growth factors such as TGF-i31, TGF-β2, and fibrinogen. Therefore, platelets, a growth factor such as TGF-i31, and fibrinogen can be prepared, and a mixture thereof can be used as the PRP in the present invention. Platelets, various growth factors, and fibrinogen can be prepared by a known method or commercially available. On the other hand, PRP is particularly rich in platelets and has a high fibrinogen content.Therefore, instead of PRP, platelets, fibrinogen, or platelets and fibrinogen are used to form bone or periodontal tissue. It is expected that a bone regenerating effect can be obtained even when the composition for use is constituted. From this, it is possible to construct the bone or periodontal tissue forming composition of the present invention by using platelets, fibrinogen, or platelets and fiprinogen instead of PRP. In addition, fibrin can be used instead of fibrinogen here. That is, for example, the composition of the present invention can be constituted by containing fibrin glue instead of PRP.

The degree of fluidity during use of the composition of the present invention is not particularly limited, and may be a gel, a slurry, a paste, a clay, a high-viscosity fluid, or the like. Preferably, it is in the form of a gel or a paste. Possible by gel or paste A composition for forming bone or periodontal tissue having excellent plasticity is obtained. Therefore, it can be applied without being previously formed into the shape of the application portion. That is, application to the application section can be easily performed. In addition, a composition for forming a bone or a periodontal tissue having a good fixation property in an application portion is obtained.

Further, it is preferable that the fluidity is such that it can be injected using an injection container at the time of use. With such fluidity, application to the application section becomes even easier. In addition, desired fluidity can be obtained depending on the application section. For example, when injecting under the periosteum, it is preferable to make the liquid more fluid (a state having a lower viscosity).

The composition of the present invention only needs to have fluidity at least at the time of use, and may be in the form of powder or solid before use. Therefore, the composition of the present invention can be in a frozen state. Further, the composition of the present invention can be used in a lyophilized state. By making the frozen or lyophilized state before use, long-term storage becomes possible, and handling before use becomes easy. Furthermore, since the antigenicity can be expected to be reduced by the freeze treatment or the freeze-drying treatment, the safety when using the same kind of PRP instead of the own PRP is improved. Although the kind of the inorganic bioabsorbable material in the present invention is not particularly limited, tricalcium phosphate (hereinafter, referred to as “—TCPJ”), α-tricalcium phosphate (hereinafter, referred to as “α-TCPJ”), tetracalcium phosphate A material selected from the group consisting of octacalcium phosphate, and amorphous calcium phosphate can be used, and these materials can be used alone, or a combination of two or more arbitrarily selected materials can be used. Preferably, either i3-TCΡ or α-TCΡ, or a combination of these in any proportion is used. More preferably,) 3-TCP is used as the inorganic bioabsorbable material. .

The inorganic bioabsorbable material can be obtained by a known method. In addition, commercially available inorganic bioabsorbable materials can also be used. jS—For TCP, for example, Olympus Optical Industrial Co., Ltd. can be used.

The inorganic bioabsorbable material is in the form of powder having a particle size such that the composition for forming bone or periodontal tissue of the present invention (hereinafter, referred to as “the present invention composition”) becomes fluid when used. Is preferred. -The powdered inorganic bioabsorbable material can be prepared by crushing and pulverizing an inorganic bioabsorbable material processed into an appropriate size to a desired particle size. The average particle size of the inorganic bioabsorbable material is 0.5! It is preferably set to 50 / m. More preferably, an inorganic bioabsorbable material having an average particle diameter of 0.5; tim to 10 / m is used. Still more preferably, the average particle diameter is 1 t π! Use an inorganic bioabsorbable material of ~ 5 // m. It is also possible to use a combination of a plurality of types of inorganic bioabsorbable materials having different particle diameters.

The inorganic bioabsorbable material preferably contains 30% by weight to 75% by weight based on the whole composition of the present invention.

The fluidity of the composition of the present invention can be adjusted by the particle size and content of the inorganic bioabsorbable material, and a desired fluidity can be obtained by appropriately adjusting both. In addition, when a gelling material and / or a thickener described below are added, the fluidity can be adjusted also by the amount of these added.

The composition of the present invention may further comprise a gelling material. For example, the composition of the present invention can be constituted by adding thrombin and calcium chloride. By adding them, thrombin acts on fibrinogen in PRP to generate fibrin. Then, the viscosity increases due to the aggregation action of fibrin. The type of the gelling agent is not particularly limited, and those which act on the components in the PRP to increase the viscosity as described above or those which exert a thickening effect by themselves can be appropriately selected and used.

In addition to the above gelled material, the composition of the present invention acts after application (after transplantation) 9800

A second gelling material that changes the fluidity (viscosity) of No. 12 can also be used in combination. With such a configuration, transplantation is easy because of appropriate fluidity at the time of use, and fixation at the application site is improved by increasing the viscosity after application, and bone or periodontal tissue Repair or regeneration can be performed effectively. In addition, there is no need to pre-mold into the shape of the application part, so versatility is high.

As the gelling material, a material having high biocompatibility is preferably used. In addition to the above-mentioned examples, collagen or fipurin glue can be used. Although various collagens can be selected and used, it is preferable to use collagen suitable for the application purpose (application tissue) of the composition of the present invention. For the purpose of regenerating bone tissue, for example, type I collagen can be used. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

The flowability of the composition of the present invention can be adjusted by adding a thickener. As the thickening agent, thickening polysaccharides such as sodium alginate, glycerin, petrolatum, etc. can be used.However, from the viewpoint of safety and Z or bone formation ability, it has high biocompatibility and is bioabsorbable or bioabsorbable. It is preferable to use a decomposable material. By adding glycerin and the like, the effect of preventing frost damage can be obtained.

For example, by employing alginate (alginate), it is possible to constitute a composition containing alginate, cells having bone forming ability, and PRP. As a result, a good bone regeneration effect is observed (see Examples below).

The composition of the present invention may contain an aqueous solvent. That is, in the first aspect of the present invention, at least PRP and an inorganic bioabsorbable material may be mixed in an aqueous solvent. In the second aspect of the present invention, at least PRP and cells having osteogenic ability are mixed in an aqueous solvent. It may be configured. As the aqueous solvent, sterile water, physiological saline, a buffer solution such as a phosphate solution and the like can be used.

The composition of the present invention may contain, in addition to the above components, a stabilizer, a preservative, a pH adjuster, and the like. It can also contain growth factors, especially osteoinductive factors (BMP).

The cells having osteogenic ability refer to cells capable of forming bone tissue, and include osteoblasts, preosteoblasts, mesenchymal stem cells that have acquired the ability to differentiate into osteogenic cells, and the like. These cells may be used in any combination. Preferred: Uses cells that have acquired the ability to differentiate into osteogenic cells as the cells having the ability to form bone. Here, “J that has acquired the ability to differentiate into bone cells refers to a state in which an undifferentiated state has been oriented to differentiate into bone cells.

As the mesenchymal stem cells that have acquired the ability to differentiate into bone cells, not only autologous cells but also allogeneic allogeneic cells can be used. In particular, cells derived from human mesenchymal stem cells can be used.

Mesenchymal stem cells that have acquired the ability to differentiate into bone cells (hereinafter referred to as “osteoblast-acquiring cells”) convert undifferentiated mesenchymal stem cells (MSCs) into bone cells. Can be prepared by culturing the cells under conditions that induce differentiation. For example, by culturing undifferentiated mesenchymal cells in a medium containing β-glycerophosphate (Dexamethason) and L-ascorbic acid (L-ascorbic acid), Differentiation can be induced. Of course, the culture conditions are not limited to those described above, and known conditions for inducing differentiation into bone cells can be employed.

Sources of undifferentiated mesenchymal stem cells include bone marrow, periosteum, pulp, and cord blood. After collecting them according to a conventional method, undifferentiated mesenchymal cells are selected based on the presence or absence of adhesiveness. In other words, select cells that have adhesive properties from cells contained in bone marrow, etc. As a result, undifferentiated mesenchymal stem cells can be obtained.

When the cells having bone differentiation potential are included, the present invention composition can be constituted by adding the extracellular matrix of the cells. This is because the extracellular matrix is expected to serve as a scaffold for the cells having the ability to acquire bone differentiation at the site to which the composition of the present invention is applied, and it is considered that the cells having the ability to acquire bone differentiation can be easily fixed at the site to which the composition is applied. In addition, it also serves as a scaffold for bone cells existing around the application site, so that high bone inducing ability can be expected. In addition, BMPs and other factors contained in the extracellular matrix are included in the composition of the present invention, and the cells having the ability to differentiate into osteogenic cells themselves, and the bone cells around the application site or stem cells having the ability to differentiate into osteogenic cells. It is expected to promote growth, proliferation and differentiation. Here, the extracellular matrix is a matrix (matrix) that surrounds the cells having the osteogenic potential.

Not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells of the same type can be used. This is because BMPs and the like contained in such extracellular matrix are of the same species, so that the same effect can be expected even when allogeneic cells are used. As described above, the fact that not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells can be used facilitates the preparation of the extracellular matrix of cells having the osteogenic differentiation potential.

When the extracellular matrix is added, the cells with the ability to acquire bone differentiation potential added simultaneously may not be living cells. This means that it is not necessary to treat the cells with the ability to acquire bone differentiation potential in the state of living cells, which is desirable from the viewpoint of handling. For example, after obtaining cells having bone differentiation potential, those obtained by freeze-drying or the like are prepared, and these can be used as cells having bone differentiation potential and their extracellular matrix.

The content of the bone based differentiation capacitation cells in the compositions of the present invention, the composition is preferably be 1 X 1 0 ⁵ or more cells are present in lml, more preferably ^{1 X 1 0 6 ~ 1 X} 1 it is preferable that 0 ⁷ cells are present. This is because by setting such a cell content, bone formation can be effectively induced. Also in the first aspect of the present invention, a composition for forming bone or periodontal tissue can be constituted by containing the above-mentioned cells having bone forming ability. In this case, the composition may be constituted by adding the extracellular matrix (extracellular matrix) of the cells having the ability to obtain bone differentiation. The composition of the present invention is prepared by mixing the above components. As a first aspect of the present invention, a method for preparing a gel composition by mixing PRP,) 3-TCP (inorganic bioabsorbable material), and thrombin and calcium chloride (gelling material) An example is shown below. First, PRP is prepared by the method described above, and / 3-TCP is added thereto and mixed. Then, add a mixture of calcium chloride solution and thrombin, and stir well with air in the syringe.

When the composition of the present invention is prepared in a frozen state or a lyophilized state, the composition has a desired fluidity when used. In the case of a frozen state, it is returned to the state before freezing by thawing. At this time, a desired fluidity can be adjusted by adding physiological saline or the like. On the other hand, in the case of a freeze-dried state, a solvent such as physiological saline is added to obtain a fluidity before the freeze-drying treatment or a state having a desired fluidity.

By enclosing the composition of the present invention in an injection container, an injection for bone or periodontal tissue formation (hereinafter referred to as “the injection of the present application”) can be obtained. For example, the composition of the present invention prepared in a fluid state is sealed in an injection container, and then frozen or freeze-dried to obtain the injection of the present invention. By using such an injection, handling becomes easier. That is, a desired effect can be expected by injecting the present injection transdermally or transmucosally without making an incision in the skin or mucous membrane as in the prior art at the time of application. Such an injection of the present invention is used after the bone or periodontal tissue forming composition enclosed therein is brought into a state having desired fluidity similarly to the above. The type of injection container is not particularly limited, and for example, a commercially available syringe can be used.

Hereinafter, examples according to the first aspect of the present invention will be described. [Example 1] Collection of PRP

(1-1) Blood collection from egret

After giving general anesthesia by administering pentobarbital from the pinna vein of four of the four Japanese white herons weighing 3.1-3.3 kg, 9 ml of blood was injected through the pinna with a 20 cc sterile syringe (manufactured by Terumo Corporation). Collected. Next, 0.5 ml of a 3.8% sodium citrate solution was placed in each of two vacuum blood collection tubes (4.5 cc, manufactured by Terumo Corporation), and 9 ml of the collected blood was divided into 4.5 ml and injected, and stored at room temperature. did. In this way, a blood sample was obtained for each egret.

(1-2) Separation of PRP

Each preserved blood sample was transferred to a 10 ml centrifuge tube, and centrifuged at 5,400 rpm for 5 minutes using a centrifuge. The centrifuged blood separated into three layers depending on the density. In order from the upper layer, there are three layers: Platelet-poor Plasma (Platelet-free plasma, PPP), Platelet-rich Prasma (PRP), and red blood cells. The top PPP (approximately 5.5 ml) was removed using a micro-mouth pipet. Next, the remaining blood components (about 3.5 ml) other than PPP were centrifuged at 2,400 rpm for 5 minutes using a centrifuge. By this operation, red blood cells and PRP were separated. The upper layer was PRP, which was collected using a micropit. About 0.35 ml of PRP was finally collected from each blood sample (9 ml).

[Example 2] Measurement of platelet count in PRP

Japanese white heron weighing 3.1-3.3 kg 24 Pentobarbital was administered from the auricular vein of 4 animals to perform general anesthesia, and then collected from the auricle using 9 ml of blood, each with a 20 cc sterile syringe (manufactured by Terumo Corporation). did. 2 Four of was measured number of platelets contained in the blood collected from a 1 two animals, an average 43.4 10 ⁴ Bruno !! 1111 ^3. Further, by using the collected from the remaining 1 2 animals blood was similarly prepared PRP as in Example 1, an average ^{457. δ Χ ΙΟ 4 / ^ !!!} 3 was measured number of platelets in PRP . From this result, the end of the egret It was confirmed that the PRP prepared in Example 1 contained much more platelets than the peripheral blood.

[Example 3] Preparation of osteogenic composition (PRP gel containing / 3-TCP)

(3-1) Preparation of i3-TCP powder

/ 3-TCP (Olympus Optical Co., Ltd., porosity 90%) was mechanically pulverized to adjust the particle diameter to about 2 / im on average. The thus prepared / 3-TCP powder was autoclaved.

(3-2) | Preparation of PRP gel containing 8-TCP

0.175 g) 3-TCP powder was placed in a 2.5 ml syringe (manufactured by Terumo Corporation). 35 ml of the PRPO prepared in Example 1 was aspirated with a micropipette; injected into a syringe containing 8-TCP powder; and mixed with) 3-TCP powder. After stirring well in a syringe, j8-TCP was dissolved in PRP. On the other hand, a mixture of 10 ml of 10% calcium chloride solution and 10,000 units of bovine thrombin (GEN-TRAC) for topical use was prepared, and 0.035 ml of this mixture was added to a syringe containing 3-TCP and PRP. Mix with O.lcc air in syringe. This initiated coagulation and formed a / 3-TCP containing PRP gel.

[Example 4] Bone formation using PRP gel containing / 3-TCP

(4-1)) 3-Space for transplanting PRP gel containing TCP

Twenty-four (24) Japanese white egrets from which blood was collected in Example 1 were shaved. After shaving, the remaining hair in the shaved area was completely removed with a depilatory cream. Next, the heron was fixed on the stomach, and the surgical field was disinfected with a chlorhexidine dalconate swab. After that, a 10 mm length skin incision was made in the upper part, and the mucosa and periosteum were peeled off to expose the bone surface of the anterior wall of the maxillary sinus. Then, the bone of the anterior wall of the maxillary sinus was removed with a diameter of about 7 mm using a dental diamond blade. Finally, the maxillary sinus mucosa was carefully dissected upward using an exfoliator to form a space for transplantation of the PRP gel containing / 3-TCP. (4-1-2)) 3—Transplantation of TCP-containing PRP grey

Obtained in Example 3; 0.175 ml of PRP gel containing 8-TCP was weighed and collected by using a 5 mm × 5 mm × 7 mm homemade space maker and implanted into the space of the right maxillary sinus just created. On the left side, a saline solution was used to give the same viscosity as i3-TCP containing PRP gel.) 3-TCP paste was transplanted. After the mucosa and periosteum were returned to their original positions, the wound was closed with suture, and the transplantation of the 3-TCP-containing PRP gel was completed.

(4-3) Evaluation of bone formation

The bone formation was evaluated by visually observing the transplanted part after a predetermined period. First, at 2 weeks and 4 weeks after transplantation, the heron transplanted at (4-2) was sacrificed, and the upper jaw and nose were removed in one lump. The extract was fixed with a 10% phosphate buffered formalin solution and decalcified with a 10% formic acid aqueous solution. Thereafter, paraffin embedding was performed, a frontal section having a thickness of 5 mm was prepared, stained with hematoxylin eosin, and observed under an optical microscope.

1) PRP group (right side of maxillary sinus transplanted with PRP gel containing () 3-TCP)

2 weeks after transplant

Figure 1 shows the transplantation site two weeks after transplantation. J3-TCP was accumulated and filled in the transplanted bone, and a large number of osteoblasts were observed to surround it. Osteoblasts were also observed in the accumulated / 3-TCP. A small amount of new bone was found around some β-TCP. Platelets were observed throughout the implanted bone. Thus, it was observed that bone formation was progressing as compared with the control group.

4 weeks after transplant

Figure 2 shows the state of the transplantation site four weeks after transplantation. 3) TCP decreased compared to 2 weeks after transplantation. It was observed that osteoblasts were encapsulated all around J3-TCP. In addition, new bone formation was observed around J3-TCP, and 2 weeks after transplantation Than had increased. Many osteoblasts also existed around the new bone. In addition, a cement line was found in the new bone. Thus, remarkable bone formation was observed as compared with the control group.

2) Control group (left maxillary sinus transplanted with i3-TCP only)

2 weeks after transplant

Figure 3 shows the state of the transplantation site two weeks after transplantation. i3-TCP was present in the transplanted bone as a square, and) osteoblasts were observed around 3-TCP. In addition, new bone formation was slightly observed.

4 weeks after transplant

Figure 4 shows the transplantation site four weeks after transplantation. Many -TCPs were still observed in blocks at the implant, and new bone formation was observed around them. A cement line was found in the trabeculae of the new bone.

(4-4) Evaluation of bone formation 8 weeks after transplantation

Eight weeks after transplantation of the i8-TCP-containing PRP gel, bone formation was evaluated again. The evaluation method was the same as in the above-mentioned cases 2 and 4 weeks after transplantation.

1) PRP group-TCP-containing PRP gel implanted on the right maxillary sinus)

8 weeks after transplant

Fig. 5 shows the state of the 3-RP containing PRP gel transplantation site (8 weeks after transplantation). Figure 5 shows a hematoxylin-eosin stained image (decalcified H.E. stained tissue image). Increased bone mass is noted at and around the implant.

Figures 6 and 7 show enlarged (16x) parts of Fig. 5. 3) The formation of new bone (B) is observed so as to be in contact with the 3-TCP-containing PRP transplant (A). That is, formation of new bone is observed in a state where no fibrous connective tissue is interposed between the transplanted portion and the new bone. This suggests that the components contained in the transplanted j3-TCP-containing PRP gel clearly induced strong osteogenesis. Thus, i3-TCP containing PRP gel It has been shown that the transplantation of osteoporosis actively forms new bone, ie, osteoinduction.

2) Control group (left maxillary sinus transplanted with i3-TCP only)

8 weeks after transplant

Figure 8 shows the state of the / 3 -TCP gel transplantation site 8 weeks after transplantation. Figure 8 shows the hematoxylin-eosin stained image (decalcified H.E. stained tissue image). There is almost no increase in bone mass compared to 4 weeks after transplantation.

Figure 9 shows an enlarged (16x) view of a portion of Figure 8. ) It is observed that the fibrous connective tissue (c) surrounds the 3-TCP gel implant (a). That is, fibrous connective tissue (c) is interposed between the transplanted part (a) and the bone (b). Therefore, it is considered that the bone observed in this photograph is not the new bone formed by the induction of (3-graft) induced by 3-TCP, but the bone metabolism originally formed by the living body.

As described above, the state of osteogenesis between the PRP group and the control group became more remarkably different at the 8 weeks after transplantation. It was also clear that strong bone induction was performed in the PRP group.

FIG. 10 is a graph summarizing the ratio (%) of the ossified portion to the whole transplanted portion. The amount of newly formed bone and the amount of newly formed bone at 2, 4 and 8 weeks after transplantation are shown. -Shows the remaining amount of TCP. In the figure, (+) indicates the results for the PRP group, and (-) indicates the results for the control group. At 8 weeks after transplantation, the amount of new bone in the PRP group was significantly higher than that in the control group. It can also be seen that the PRP group absorbed) 3-TCP better.

Next, an example according to the second aspect of the present invention will be described.

[Example 5] Collection of PRP

Before transplantation, blood was collected from two dogs weighing 15-17 kg, and treated in the same manner as in Example 1. PRP was separated and purified. Each blood sample (50 ml) resulted in approximately 5 ml of PRP each. The number of platelets in the PRP was counted in the same manner as in Example 2, and it was confirmed that the platelets were contained abundantly.

[Example 6] Preparation of bone marrow cells

(6-1) Removal of bone marrow fluid from dog iliac bone

The iliac bone marrow fluid was collected from a dog iliac bone using a bone marrow puncture needle into a lOcc syringe (manufactured by Terumo Corporation) and injected into a centrifuge tube containing a heparin-containing basic medium.

(6-2) Seeding bone marrow cells

An appropriate amount (concentration: 30%) of the basal medium was added to the bone marrow suspension collected in the centrifuge tube, and the bone marrow suspension was seeded on a flask (80 cm ² , Greiner labortec nik Germany). (6-3) Selection and culture of adherent cells (undifferentiated mesenchymal stem cells)

The flask seeded with bone marrow fluid transferred into Inkyubeta were cultured under conditions of _{5% C0 2, 37 ° C} . The medium was replaced 24 hours after seeding the bone marrow cells in order to remove the blood cells contained in the culture medium and select and adherent cells for culture. Thereafter, culturing was continued for about 7 days in the culture medium until the cells became subconfluent. During that time, medium exchange was performed once every three days.

(6-4) Selection and culture of adherent cells (undifferentiated mesenchymal stem cells)

The flask seeded with bone marrow fluid was transferred into Inkyube Isseki were cultured under conditions of 5% C0 _2, 37. The medium was replaced 24 hours after seeding the bone marrow cells in order to remove the blood cells contained in the culture medium and select and adherent cells for culture. Thereafter, culturing was continued for about 7 days until the cells became subconfluent in the culture solution. During that time, the medium was changed once every three days. In addition, MSCGM, 50 U / ml penicillin G, and 50 pg / ml streptomycin (manufactured by Poietics) were added to the culture solution.

Subsequently, the cells were detached using 0.05% trypsin, inoculated so that the area ratio became 2 to 4 times, and subcultured. (6-5) Induction of differentiation of undifferentiated mesenchymal stem cells

After culturing for about 6 days from the passage, confirm that the cells have reached subconfluence. Culture medium Pico | 3-glycerophosphate Sigma Chemical Co., St Louis, US), Dexamethasone (Sigma Chemical Co., St Louis , USA), and VitaminC phosphate (L-ascorbic acid phosphate magnesium salt n-hydrate, Sigma Chemical Co., St Louis, USA) to 10 mM, 10.8 M, and 0.05 mM, respectively. (Bone induction medium) The culture was continued. The medium was replaced every three days, and | 3-glyceropnosphate, Dexamethasone, and VitaminC phosphate were added to the culture medium in the same manner as above.

(6-6) Recovery of cell components

The medium was removed from the flask, leaving a small amount of medium. The cells were detached from the flask using Trypsin / EDTA. The detached cells were transferred to a centrifuge tube (FALCON ECTON DICKINSON USA) together with the medium, and subjected to centrifugation (1500 rpm, 5 min). The supernatant was aspirated and the cell components were collected.

[Example 7] Preparation of osteogenic composition (PRP-cell-containing gel)

The cell component recovered in Example 6 was suspended in a small amount of PRP to prepare a cell suspension. 3.5 ml of the PRP obtained in Example 5, 0.5 ml of a 10% calcium chloride solution, and topical bovine thrombin (0000 units of GEN-TRAC) were mixed in a syringe to form a PRP-cell-containing gel.

On the other hand, a gel (PRP gel) prepared by mixing PRP, a 10% calcium chloride solution, and topical bovine thrombin was prepared as a control.

[Example 8] Bone formation using PRP-cell-containing gel

(8 — 1) Creating transplant space

The dog from which blood was collected in Example 5 was fixed on the stomach, and the surgical field was disinfected with a chlorhexidine dalconate swab. Then, extract the teeth one month beforehand A skin incision about 100 mm in length was made in the jaw, and the mucosa and periosteum were peeled off to expose the bone surface of the jawbone. Then, using a trephine pad, a jaw bone defect was created with a size of about 10 mm.

(8-2) Transplantation of PRP-cell-containing gel

About 4 ml of the PRP-cell-containing gel prepared in Example 7 was injected into the jaw bone defect. As a control, a group in which PCBM was transplanted (autologous bone transplantation), a group in which the PRP gel obtained in Example 7 was transplanted, and a group in which only the defect was obtained (no transplantation) were prepared.

(8-3) Evaluation of bone regeneration ability (osteogenesis)

The bone regeneration ability of each group was histologically evaluated and compared by visually observing the transplanted part after a predetermined period.

First, at 2, 4 and 8 weeks after transplantation, transplanted parts were removed from dogs transplanted at (8-2). The extract was fixed with a 10% phosphate buffered formalin solution and decalcified with a 10% aqueous formic acid solution. Thereafter, tissue-stained sections were prepared by embedding in paraffin, stained with hematoxylin and eosin, and observed under an optical microscope.

1) PCBM group (autologous bone transplantation)

Figure 11 shows the two weeks (2 W), four weeks (4 W), and eight weeks (8 W) after transplantation of the PCBM transplant. Two weeks after transplantation, it is observed that bone regeneration has already taken place. At the same time, absorption of the transplanted PCBM is also observed. Eight weeks after transplantation, good bone regeneration was obtained up to the margin of the defect (arrow E in the figure).

2) PRP-cell group (PRP-cell-containing gel transplant group)

Fig. 12 shows the results of two weeks (2 W), four weeks (4 W), and eight weeks (8 W) after transplantation of the PRP-cell-containing gel transplant. It was observed that the amount of new bone (arrow F in the figure) increased over time after transplantation, and good bone regeneration was performed up to the margin of the defect (arrow G in the figure) at 8 weeks after transplantation. You can see it is being done. It is also observed that precise bone regeneration is being performed. Furthermore, abundant angiogenesis (arrow H in the figure) was observed, It can be seen that the formation of new bone is induced. The degree of bone regeneration at 4 weeks and 8 weeks after transplantation was similar to that of the PCBM group (Fig. 11).

3) PRP group (PRP gel implanted group)

Fig. 13 shows the state of the PRP gel transplantation site at 2 weeks (2 W), 4 weeks (4 W), and 8 weeks (8 W) after transplantation. After transplantation, bone regeneration is observed to occur gradually from the bottom of the defect (arrow I in the figure), but the degree of bone regeneration is low. Even at 8 weeks after transplantation, bone regeneration was not performed up to the defect margin (arrow J in the figure).

4) Defect only (no transplant)

Fig. 14 shows the state of the defect left without transplantation at 2 weeks (2 W), 4 weeks (4 W), and 8 weeks (8 W). As in the case of the PRP gel group, it is observed that osteogenesis occurs gradually from the bottom of the defect (arrow K in the figure). The degree of bone regeneration is extremely low. Even after 8 weeks, almost no bone regeneration has occurred at the periphery of the defect, and fibrous cells are observed to be aggregated (arrow L in the figure).

As described above, it was found that the use of the gel containing PRP-cells provided good bone regeneration equivalent to that of PCBM. In other words, it was shown that a sufficient bone regeneration effect can be obtained by the combination of injectable PRP and cells having osteogenic ability even in comparison with autologous bone transplantation (PCBM).

Next, an osteogenic ability evaluation test (Examples 9 to 12) using a composition obtained by combining fibrinogen, i3-TCP, and osteogenic cells will be described.

[Example 9] Fabrication of porous i3-TCP block

First, calcium carbonate, calcium hydrogen phosphate and water were mixed, mechanically impacted and pulverized, and mechanochemically synthesized. After that, drying and firing (900 ° C, 5 hours) were performed, and after granulation, a fluid muddy mixture was prepared. After immersion in a combustible sponge, drying and firing again (1125 ^ 0, 3 hours).

The completed porous 3-TCP block has a three-dimensional porous structure in which all pores are continuous. It was made. Using this, a cylindrical block with a diameter of about 5 mm and a height of about 4 mm was manufactured, and the pore diameter was adjusted to 200 to 400 πι and the porosity was adjusted to about 90%.

[Example 10] Collection and culture of cells

Bone marrow cells were collected from the femur of a 7-week-old male male rat. The collected bone marrow cells were cultured in Minimum Essential Medium (MEM medium) supplemented with 15% fetal calf serum and antibiotics (penicillin 1000 U / ml, streptomycin 0.1 mg / ml, amphotericin B 0.25 ^ ig / ml). Primary culture was performed for one day. Culturing was carried out at 37 ° C, 5% C0 ₂ below. Thereafter, a cell suspension was prepared by trypsin-EDTA treatment, and the cell concentration was adjusted to 10 ⁶ cells / ml.

[Example 11] Preparation of transplant composition

A cell / β-TCP complex was prepared by immersing the β-TCP block in the above cell suspension. Cells /) 3 -TCP complex the medium ^{10 · 8 Μ Dexamethasone (Sigma Chemical} Co., St Louis, USA Seo, lOmM β -glycerophosphate (Sigma Chemical Co. , St Louis, US)> 50 β g / ml VitaminC phosphate (L-ascoroic acid phosphate magnesium salt n _ hydrate> Sigma Chemical Co., St Louis, USA) were added thereto to carry out a 20-day subculture. in the culture, media exchange was performed three times a week, the In each case, 3) -glycerophosphate Dexamethasone and OitaminC phosphate were added to the culture solution.

The cultured 3-cell complex was disrupted with forceps and suspended in the culture medium. Solution A consisting of a suspension of fibrinogen (80 mg) and fibrin stabilizing factor XIII (75 units) in 1 ml of plasmin inhibitor and aprotinin (1000 kIE / ml) (Kumamoto, Japan). A suspension containing the MSCs / β-TCP complex obtained in this way and a solution B containing thrombin (250 units) dissolved in 1 ml of 53 mM CaCh solution (Kumamoto Institute for Chemistry and Serum Therapy, Kumamoto, Japan) in a sterile syringe (5 ml) To form a composition for transplantation (fibrinogen- | 3-TCP-cell-containing composition) (Solution A: Solution B = 1: 1 (volume / volume)) o The concentration of MSCs in this admixture is It was about 10 × 10 ⁶ cells / ml. By mixing the solution A and the solution B at room temperature, a coagulation action occurs to form a fibrin glue consisting of a semi-rigid three-dimensional tissue.

[Example 12] Bone formation using a composition for transplantation (fibrinogen-1) 3-TCP-cell-containing composition

(12-1) Transplantation of transplant composition

About 1.5 ml of the composition for transplantation was transplanted subcutaneously on the back of a syngeneic rat. As a control, a porous; 3-TCP block containing no bone marrow cells was crushed and mixed with fibrinogen to form a gel, which was transplanted subcutaneously on the back of a syngeneic rat (control group). (1 2-2) Evaluation of bone regeneration ability (osteogenesis ability)

The bone regeneration ability was evaluated by histological observation of the transplanted part at 2, 4 and 8 weeks after transplantation. The transplanted part was removed, fixed with 10% phosphate buffer formalin, and then decalcified with 10% formic acid aqueous solution. Thereafter, tissue staining sections were prepared by embedding in paraffin, stained with hematoxylin and eosin (H.E.), and observed under an optical microscope.

1) Group transplanted with transplantation composition (fibrinogen-i3-TCP-cell-containing composition) FIG. 15 is a photograph showing a directly observed transplantation site 8 weeks after transplantation. A white lump is observed at the transplant (arrow).

Fig. 16 shows the H.E.-stained tissue images at 2 weeks (2W), 4 weeks (4W), and 8 weeks (8W) after transplantation of the transplanted part. Two weeks after transplantation, osteoblast lining is seen (arrow), indicating that bone formation is taking place. At 4 weeks after transplantation, bone cells became visible (arrows), indicating that active bone formation was progressing. Eight weeks after transplantation, some lamellar structures are seen (arrows), and bone maturation can be observed.

2) Control group ''

FIG. 17 is a HE-stained tissue image of the control group at 8 weeks after transplantation. Figure 1 As shown in Fig. 7, the control group showed only a slight bone formation 8 weeks after transplantation.

From the above results, significant bone regeneration was observed in the group transplanted with the transplant composition (fibrinogen-1) 3-TCP-cell-containing composition as compared with the control group.

Next, an osteogenic ability evaluation test (Examples 13 to 16) using a composition obtained by combining PRP, alginate (alginate), and cells having osteogenic ability will be described.

[Example 13] Collection of PRP

PRP was isolated and purified from rat peripheral blood in the same manner as in Example 1. From each blood sample (50 ml), about 5 m of PRP was finally obtained.

[Example 14] Preparation of bone marrow cells

(14-1) Removal of bone marrow fluid from human iliac bone

The iliac bone marrow fluid was collected from a human iliac bone using a bone marrow puncture needle into a lOcc syringe (manufactured by Terumo Corporation) and injected into a centrifugal tube containing a heparin-containing basic medium.

(14-2) Seeding bone marrow cells

An appropriate amount (concentration: 30%) of the basal medium was added to the bone marrow suspension collected in the centrifuge tube, and the bone marrow suspension was seeded on a flask (80 cm2, Greiner labortechnik Germany). (14-3) Selection and culture of adherent cells (undifferentiated mesenchymal stem cells)

The flask inoculated with the bone marrow fluid was transferred into Incube overnight, and cultured under the conditions of 5% C02, 37. The medium was replaced 24 hours after seeding the bone marrow cells in order to remove the blood cells contained in the culture medium and select and adherent cells for culture. Thereafter, culturing was continued for about 7 days in the culture solution until the cells became subconfluent. During that time, medium exchange was performed once every three days.

(14-14) Selection and culture of adherent cells (undifferentiated mesenchymal stem cells)

The flask seeded with bone marrow fluid was transferred into the Inkyubeta, under conditions of 5% C0 _2, 37 ° C Cultured. The medium was replaced 24 hours after seeding the bone marrow cells in order to remove the blood cells contained in the culture medium and select and adherent cells for culture. After that, the culture was continued for about 7 days in the culture solution until it became subconfluent. During that time, the medium was changed once every three days. In addition, MSCGM, 50 U / ml penicillin G, and 50 g / ml streptomycin (manufactured by Poietics) were added to the culture solution.

Subsequently, cells were detached using 0.05% trypsin, seeded so that the area ratio became 2 to 4 times, and subcultured.

(14-5) Differentiation induction of undifferentiated mesenchymal stem cells

First, culture was performed for about 6 days from the passage, and it was confirmed that the cells reached subconfluency. USA), and VitaminC phosphate (L-ascorbic acid phosphate magnesium salt n-hydrate, Sigma Chemical Co., St Louis, USA) to 10 mM, 10 "8 M, and 0.05 mM, respectively. (Osteogenesis medium) The culture was continued The medium was changed every three days, and jS-glycerophosphate, Dexamethasone, and VitaminC phosphate were added to the culture solution each time as described above.

(14-6) Recovery of cell components

After culturing in osteoinductive medium for about 6 days, the medium was removed from the flask, leaving a small amount of medium. The cells were detached from the flask using Trypsin / EDTA. The detached cells were transferred to a centrifuge tube (FALCON ECTON DICKINSON USA) together with the medium, and subjected to a centrifugation treatment (1500 rpm, 5 min). The supernatant was aspirated and the cell components were collected.

[Example 15] Preparation of transplantation composition (PRP-alginate-cell-containing composition) The cell component recovered in Example 14 was suspended in a small amount of PRP to prepare a cell suspension. About 1.5 ml of this cell suspension and alginate (manufactured by Sigma), obtained in Example 13 About 0.15 ml of the obtained PRP was mixed in a syringe to form a composition for transplantation (PRP-arginine-cell-containing composition).

[Example 16] Bone formation using a composition for transplantation (PRP-alginate-cell-containing composition)

(16-1) Transplantation of transplant composition

About 1.5 ml of the composition for transplantation was subcutaneously transplanted to the back of a 4-week-old KSN nude mouse. On the other hand, those to which an arginite was transplanted in place of the composition for transplantation were used as a control group.

(16-2) Evaluation of bone regeneration ability (osteogenesis ability)

At 16 weeks after the transplantation, the transplanted site was observed histologically and the bone regeneration ability was evaluated. First, the transplanted part was excised, fixed with 10% phosphate buffer formalin, and then decalcified with a 10% formic acid aqueous solution. Thereafter, tissue staining sections were prepared by embedding in paraffin, stained with hematoxylin-eosin (H.E.), and observed under an optical microscope. FIG. 18 shows an H.E.-stained tissue image of 16 weeks after transplantation ((A) is a group transplanted with the composition for transplantation, (B) is a control group). In the group (A) into which the transplant composition was transplanted, new bone was observed (arrow), and it was confirmed that bone regeneration was performed. On the other hand, in the control group (B), the transplanted alginate was stained and observed (arrow), but no new bone formation was observed. It was shown that in the group transplanted with, bone regeneration was significantly performed as compared with the control group. The present invention is not limited to the description of the above embodiment and Examples. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. Industrial applicability

The composition for forming bone or periodontal tissue of the present invention can repair and regenerate bone tissue or periodontal tissue. It can be applied to various fields required. For example, it can be applied to bone augmentation when implanting artificial dental roots in a ridge with high bone resorption. In addition, the present invention can be applied to regeneration of bone tissue at a bone defect caused by trauma or various bone diseases, and reinforcement or supplementation of bone. In addition, the present invention can be applied to regeneration of alveolar bone and periodontal tissue in a defective portion of alveolar bone due to periodontal disease or the like. As an application method, a bone-forming composition such as a gel or a paste is filled, injected, or applied to the application site.

The composition for synthesizing bone or periodontal tissue of the present invention contains a large number of growth factors capable of promoting the proliferation or differentiation of cells of the osteogenic system. Therefore, the cells of the osteogenic system can be efficiently proliferated or differentiated at the application part (transplantation part), and the formation of bone tissue or periodontal tissue can be promoted. The use of autologous PRP is considered to improve the quality and quantity of bone tissue or periodontal tissue regeneration by the above non-toxic and immunoinactive growth factors. Further, since the first aspect of the present invention contains an inorganic bioabsorbable material serving as a scaffold for bone-forming cells, the growth of the bone-forming cells in the application portion is promoted, and appropriate plasticity is imparted. The form can be maintained. In addition, since it has a gel-like fluidity, it can be easily inserted into the application area using a syringe needle or the like (it is also possible to apply without opening the wound area). High versatility without the need for pre-molding. As described above, by using the composition for forming bone or periodontal tissue of the present invention, it is not necessary to collect autologous bone and subject it to transplantation, and it is possible to easily grow bone or periodontal tissue. Become.

Claims

The scope of the claims

1. A composition for forming bone or periodontal tissue, which comprises PRP and an inorganic bioabsorbable material and has fluidity during use.

2. A composition for forming bone or periodontal tissue which has fluidity during use, comprising platelets, fibrinogen, and an inorganic bioabsorbable material.

3. A composition for forming bone or periodontal tissue having fluidity during use, comprising platelets, fibrin, and an inorganic bioabsorbable material.

4. A composition for forming bone or periodontal tissue, which comprises platelets and an inorganic bioabsorbable material and has fluidity during use.

5. The inorganic bioabsorbable material is selected from the group consisting of:) 3-tricalcium phosphate, α-triphosphate phosphate, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. The composition for forming a bone or periodontal tissue according to any one of claims 1 to 4, wherein the composition is one or more inorganic bioabsorbable materials.

6. The bone or tooth according to any one of claims 1 to 5, wherein the inorganic bioabsorbable material has an average particle diameter of 0.5; tim to 50 im. Peripheral tissue forming composition.

7. The bone or periodontal tissue formation according to any one of claims 1 to 6, wherein the inorganic bioabsorbable material is contained in an amount of 30% by weight to 75% by weight. Composition.

8. The composition for forming a bone or periodontal tissue according to any one of claims 1 to 7, which is prepared further comprising cells having an osteogenic ability.

9. A composition for forming bone or periodontal tissue, which comprises PRP and cells having osteogenic ability and has fluidity during use.

10. A composition for forming bone or periodontal tissue having fluidity during use, comprising platelets, fibrinogen, and cells capable of forming bone.

1 1. A composition for forming bone or periodontal tissue which has fluidity when used, comprising platelets, fibrin, and cells capable of forming bone.

1 2. A composition for forming bone or periodontal tissue, which comprises platelets and cells having an osteogenic ability and has fluidity during use.

1 3. A composition for forming bone or periodontal tissue having fluidity during use, comprising fibrinogen, an inorganic bioabsorbable material, and cells having an osteogenic ability.

14. The inorganic bioabsorbable material is selected from the group consisting of / 3-tricalcium phosphate, α-tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, and amorphous phosphate. 14. The composition for forming bone or periodontal tissue according to claim 13, wherein the composition is one or more inorganic bioabsorbable materials.

1 5. A composition for forming bone or periodontal tissue which has fluidity when used, comprising PRP, alginate, and cells having bone forming ability.

1 6. A bone or periodontal tissue forming device according to any one of claims 1 to 15, characterized in that the device has fluidity that can be injected using an injection container at the time of use. Composition.

1 7. The composition for forming a bone or periodontal tissue according to any one of claims 1 to 16, wherein the composition further comprises a gelling material.

18. The composition for forming bone or periodontal tissue according to claim 17, wherein the gelling material is thrombin and calcium chloride.

1 9. The composition for forming bone or periodontal tissue according to any one of claims 1 to 18, wherein the composition is used after being thawed from a frozen state at the time of use.

20. An injection for forming bone or periodontal tissue, wherein the composition for forming bone or periodontal tissue according to any one of claims 1 to 19 is enclosed in an injection container.