JP2006122518A - Composition for forming bone or periodontium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a composition for use in establishing clinically applicable reconstruction (regeneration) of a bone or periodontium. <P>SOLUTION: This composition for forming the bone or periodontium is composed of a combination of platelet-rich plasma (PRP) of a predetermined concentration rate and cells with osteogenic ability. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a composition that can be used for repair or regeneration (reconstruction) of bone tissue or periodontal tissue.

Numerous studies have been conducted in the fields of craniofacial surgery, plastic surgery, and orthopedic surgery for the purpose of reconstruction and repair of bone defects. However, for both soft and hard tissues, the use and preparation of biocompatible materials remains a problem to be solved clinically. Until now, resected self-tissues including fat, fascia, and bone fragments have been frequently used. However, conventional treatment methods have individual problems. Appropriate self-materials are not sufficient for targeted reconstruction due to limited supply, susceptibility of donor sites, and sometimes poor tissue quality or difficult to mold as grafts. Even if it is other-family material, the supply amount is limited due to the shortage of donors. On the other hand, the use of artificial restorations increases the susceptibility to infection and is associated with the problem of unknown invasion to the surroundings and long-term interaction with host physiology.
As a means to solve these problems in organ-organ transplantation, Vacanti et al. (1-3) use a construct formed by combining biocompatible scaffolds and growth factors on isolated cells. He proposed a new technique called “tissue engineering” with morphogenesis of new tissue. This technique usually requires open transplantation of cell-scaffold constructs. If this technology is applied to osteoblasts and a system is developed that can be transplanted with minimally invasive cell-scaffold constructs, then the tissue engineering applicability will include, for example, craniofacial reconstruction, plastic surgery, and orthopedics. It spreads to many areas such as the surgical field.

Mesenchymal stem cells (MSCs) can be replicated as undifferentiated cells and have the ability to differentiate into mesenchymal tissue lineages including bone, cartilage, fat, tendon, muscle, and bone marrow stroma (Non-Patent Documents 4 and 5), and has attracted widespread attention because of its high potential utility in the field of tissue engineering.
So far, it has been shown that bone tissue can be repaired by using MSCs alone (Non-Patent Documents 5 and 6). However, if an attempt is made to fill the defect with only MSCs, a very large number of MSCs are required, and it is difficult to maintain MSCs in the bone defect. For these reasons, many researchers have recently attempted to use ceramics as a scaffold (Non-patent Document 7).
In the previous study, we investigated the bone-forming ability of a new bioresorbable β-triphosphate (β-TCP) combined with MSCs as a scaffold and compared it with hydroxyapatite (HA) (Non-patent Document 8). ). As a result of this study, this scaffold has the ability to form bone, but is fragile, and new bone enters bone defects due to lack of through-holes, low absorbency of ceramics, and absence of osteoinductive properties It turned out that things were disturbed. Next, we use a minimally invasive and jelly-like flexible material that can be used to deliver autologous bone to treat or reconstruct bone defects due to osteoporosis, periodontitis, and tumor resection. Tried. First, fibrin glue was used as an injectable scaffold. With the injectable MSCs / β-TCP-fibrin glue mixture, it was possible to form a three-dimensional scaffold necessary for good transplantation and engraftment of osteoblasts (Non-patent Document 9). Examples using osteoinductive materials, graft flocculants, and fibrin as a graft material have been reported (Non-Patent Documents 10 and 11). In addition, the importance of growth factors found in self-fibrin adhesives has been pointed out (Non-Patent Documents 12 to 14). We therefore considered using a platelet rich plasma (PRP) gel consisting of a mixture of growth factors and modified self-fibrin glue. PRP gels are obtained by mixing platelet-rich plasma with thrombin and calcium chloride, obtained by centrifuging autologous whole blood under a number of different conditions. The critical difference between PRP gels and fibrin glue is that platelets are present at high concentrations and that there is an original fibrinogen concentration. Platelets, once activated in the presence of thrombin, form a scaffold by releasing a myriad of factors and forming a fibrin clot. Using PRP has several advantages, such as enhanced and accelerated bone regeneration, faster and predictable soft tissue healing. Receptors for transforming growth factor β1 (TGF-β1), transforming growth factor β2 (TGF-β2), and platelet-derived growth factor (PDGF) have been found in medullary bone through the use of monoclonal technology (non-patented) References 12-14). Studies using monoclonal antibodies show that high concentrations of PDGF and TGF-β are present in the plasma, so that these factors are present in the original plasma used to obtain self-fibrin. Proved.
In addition, the technique relevant to this application is disclosed by the international publication 02/17983 pamphlet and the international publication 02/40071 pamphlet (patent documents 1, 2).

International Publication No. 02/17983 Pamphlet International Publication No. 02/40071 pamphlet Langer, R., and Vacanti, J.P.Tissue engineering.Science 260, 920, 1993. Vacanti, J.P., Morse, M.A., Saltzman, W.M., Domb, A.J., Perez-Atayde, A., and Langer, R. Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg 23, 3, 1988. Vacanti, J.P. Beyond transplantation. Arch. Surg 123, 545, 1998. Pittenger, MF, Mackay, AM, Beck, CB, Jaiswal, RK, Douglas, R., Mosca, JD, Moorman, MA, Simonetti, DW, Craig, S., and Marshak, DR Multilineage potential of adult human mesenchymal stem cells Science 284, 143, 1999. Owen, M., and Friedenstein, A.J.Stromal stem cells: Marrow derived osteogenic precursors.Ciba Found Symp 136, 42, 1998. Kadiyala, S., Young, R.G., Thiede, M.A., and Bruder, S.P.Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro.Cell Transplant 6, 125, 1997. Bruder, SP, Kurth, AA, Shea, M., Hayes, WC, Jaiswal, N., and Kadiyala, S. Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells.J Orthop Res 16, 155, 1998 . Boo, JS, Yamada, Y., Hibino, Y., Okazaki, Y., Okada, K., Hata, K., Yoshikawa, T., Sugiura, Y., and Ueda, M. Tissue-engineered bone using mesenchymal stem cells and a biodegradable scaffold. J Craniofac Sug 13, 231, 2002. Yamada, Y., Boo, JS, Ozawa, R., Nagasaka, T., Okazaki, Y., Hata, K., and Ueda, M. Bone regeneration following injection of mesenchymal stem cells and fibrin glue with a biodegradable scaffold. J Cranio-Maxill Sug 31, 27, 2003. Rosenberg, M., and Mortenson, W. Considerations in the corticosteroid treatment of bone cysts. J Pediatr Orthop 9, 240, 1989. Schlag, G., and Redl, H. The influence of fibrin sealant on demineralized bone matrix-dependent osteoinduction: A quantitative and qualitative study in rats.Clin Orthop 238, 282, 1989. Bowen-Pope, DF, Vogel, A., and Ross, R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents.Pnas 81, 2396, 1984. Roberts, A.B., and Spron, M.B.Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta) .Growth factors 8, 1, 1993. Antonaides, H.N., and Williams, L.H.Human platelet-derived growth factors: Structure and functions. Federation Proc 42, 2630, 1983. Wada, K., Niimi, A., Watanabe, K., Sawai, T., and Ueda, M. Maxillary sinus floor augmentation in rabbits: a comparative histologic-histomorphometric study between rhBMP-2 and autogenous bone.Int J Periodontics Restorative Dent 21, 253, 2001. Kadiyara, S., Jaiswal, N., and Bruder, S. Culture-expanded bone marrow-derived mesenchymal stem cells can regenerate a critical-size segmental bone defect.Tissue Engineering 3, 173, 1997. Petite, H., Viateau, V., Bensaid, W., Meunier, A., de Pollak, C., and Bourguignon, C. Tissue-engineered bone regeneration.Nature Biotech 18, 959, 2000. Haynesworth, S.E., Goshima, J., Goldberg, V.M., and Caplan, A.I.Characterization of cells with osteogenic potential from human marrow.Bone 13, 81, 1992. Bruder, S.P., Jaiswal, N., and Haynesworth, S.E.Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem 64, 278, 1997. Marks, S.C., and Hermey, D.C.The structure and development of bone.In: Bilezikian JP, Raisz LG (ed) Principles of bone biology.Academic Press, San Diego, CA, 1996, pp3-14. Zawicki, D.F., Jain, R.K., Schmid-Schoenbein, G.W., and Chien, S. Dynamics of neovascularization in normal tissue.Microvasc Res 21, 27, 1981. Mundy, GR, Rodan, SB, Majeska, RJ, DeMartio, S., Trimmier, C., Martin, TJ, and Rodan, GA Unidirectional migration of osteosarcoma cells with osteoblast characteristics in response to products of bone resorption.Calculated Tissue International 34 , 542, 1982. Hill, P.A., and Orth, M. Bone remodeling.Br J Orthodontics 25, 101, 1998. Marx, R.E., Carlson, E.R., Eichstaedt, R.M., Schimmele, S.R., Stauss, J.E., and Georgeff, K.R.Platelet-rich plasma, growth factor enhancement for bone grafts.OS OM OP 85, 638, 1998. Soslau, G., Morgan, D.A., Jaffe, J.S., Brodsky, I., and Wang, Y. Cytokine mRNA expression in human platelets and a megakaryocytic cell line and cytokine modulation of platelet function.Cytokine 9, 405, 1997. Khouri, R.K., Koudsi, B., and Reddi, H. Tissue transformation into bone in vivo. JAMA 266, 1953, 1991. Kaban, L.B.Calvarial bone regeneration using osteogenin.J Oral Maxillofac Surg 47, 1187, 1989.

  In view of the above background, the present invention provides a composition used for establishing a bone or periodontal tissue reconstruction (regeneration) technique that can be applied clinically, and a use form (such as a reconstruction method) of the composition. The purpose is to provide.

In view of the above object, the present inventors have found that platelet rich plasma (PRP) has the potential to promote new bone formation, is harmless, does not undergo immune rejection, and further wound healing. Focused on activating. Therefore, we promoted bone formation using PRP as a self-scaffold (scaffold) combined with mesenchymal stem cells (MSCs) prepared in vitro. In addition, the osteogenesis promoting effect was compared with the case where the scaffold alone was used as a comparative control (PRP group), and the case where autoclastic cancellous bone and bone marrow (PCBM) were used (PCBM group). Radiation amount, histological analysis, and histomorphometric analysis were used for evaluation of new bone. As a result of histological observation, the group using a combination of canine MSCs and PRP (dMSCs / PRP group) compared with the control group (deletion only), PRP group, and PCBM group at 2 weeks and 4 weeks after transplantation ) Showed good mature bone formation and angiogenesis. As a result of histomorphometric evaluation, the area of new bone in each group after 8 weeks of treatment was 18.3 ± 4.84% (control group), 29.2 ± 5.47% (PRP group), 61.4 ± 3.38% (PCBM group), and It was 67.3 ± 2.06% (dMSCs / PRP group), and a significant difference was recognized among the PCBM group, the dMSCs / PRP group, and the control group.
From these results, it was shown that a mixture of mesenchymal stem cells and PRP is extremely useful as a material for bone regeneration (reconstruction). On the other hand, focusing on the platelet concentration rate (platelet concentration) of the PRP used, and examining this, it was found that the bone regeneration effect depends on the platelet concentration rate (platelet concentration). That is, it was found that the use of PRP having a predetermined platelet concentration rate (platelet concentration) is important for obtaining a high bone regeneration effect.
The present invention has been completed based on the above findings, and provides the following configurations.
That is, the present invention provides a composition for forming bone or periodontal tissue, comprising platelet-rich plasma (PRP) having a platelet concentration rate in the range of about 150% to about 1500% and cells having osteogenic potential. It is. As the cells having bone forming ability, mesenchymal stem cells induced to differentiate into bone cells are preferably used.
In another aspect of the invention, the composition is composed of platelet rich plasma having a platelet concentration in the range of about 300% to about 600%.
In one embodiment of the present invention, the platelet concentration of the composition is in the range of about 200,000 / μL to about 6,150,000 / μL.
In a further aspect of the present invention, there is provided a composition for forming bone or periodontal tissue substantially composed only of platelet-rich plasma (PRP) and cells capable of osteogenesis.
In one aspect of the present invention, a composition for forming a bone or periodontal tissue is provided, which further comprises a gelling material. As the gelling material, thrombin and calcium chloride can be used.
The composition of the present invention preferably has fluidity that can be injected using an injection container at the time of use. The composition of the present invention can be used after being frozen once and then thawed from the frozen state at the time of use.
The composition of the present invention can be provided for use in dental implants, periodontal diseases, cysts, extraction sockets, bone extensions, or cleft bone graft sites.
As another form of the present invention, there is provided an injection for forming bone or periodontal tissue, wherein the composition is sealed in an injection container.

By applying the composition of the present invention, good bone regeneration (reconstruction) is promoted in a bone (or periodontal tissue) defect portion. Specifically, by containing PRP containing abundant growth factors that have bone cell proliferation and differentiation promoting effects, these growth factors enable effective bone tissue (or teeth) at the application site (transplant). Regeneration of the surrounding organization can be expected. Moreover, in the composition of this invention, the reproduction | regeneration of the positive bone tissue (or periodontal tissue) by the said cell can be anticipated by containing the cell which has bone formation ability.
Furthermore, according to the above configuration, since it is possible to prepare the transplant material in a fluid state at the time of use, there is no need to mold it in advance according to the shape of the bone (or periodontal tissue) defect part. It is easy to handle.
In addition, according to the present invention, since a bone (or periodontal tissue) defect can be repaired and reconstructed without collecting autologous bone, the burden on the patient associated with the treatment can be reduced.
Thus, the composition of the present invention is extremely useful as a material for bone (or periodontal tissue) regeneration.

In the present invention, a composition for forming bone or periodontal tissue is constructed by combining platelet-rich plasma (PRP) and cells having osteogenic ability.
Here, “platelet-rich plasma” is PRP (Platelet-rich Plasma), that is, plasma rich in platelets. In other words, it refers to plasma in which platelets are concentrated. PRP is prepared, for example, by subjecting the 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 contain abundant growth factors such as Platelet-derived Growth Factor (PDGF), Transforming growth factor β1 (TGF-β1), Transforming growth factor β2 (TGF-β2) (Jarry J. Peterson : Oral surg Oral Med Oral Pathol Oral Radiol Endod, 85, 638-646 (1998)).

Here, if an example of the preparation method of PRP is shown, first, anticoagulants such as sodium citrate will be added to the collected blood and left at room temperature for a predetermined time. Thereafter, centrifugation is carried out under conditions that separate blood cells and buffy coat (for example, about 54,000 rpm). Thereby, it is separated into two layers (the upper layer is called Platelet-poor Plasma. The lower layer includes blood cells and buffy coat). After removing the upper layer, it is further centrifuged under conditions that separate red blood cells (for example, about 2,400 rpm). The fraction (Platelet-rich Plasma: PRP) substantially free of erythrocytes obtained as a result is collected. The preparation method of PRP is not limited to the said method, It can prepare by the method which added correction as needed.
Preferably, autologous PRP is used. This eliminates the risk of toxicity or immune rejection.

Although there is no general definition for the number of platelets contained in PRP (platelet concentration rate), plasma containing about 150 to about 1500 times platelets compared to the collected blood is used as PRP in the present invention. Can do.
In the present invention, the “platelet concentration rate” of PRP is represented by the following formula.
Platelet concentration rate (%) = (average platelet count in PRP / average platelet count in whole blood as starting material) x 100
Therefore, for example, when the average platelet count in PRP is 1,000,000 and the average platelet count in whole blood is 300,000, the platelet concentration rate is about 3333%. As shown in the following examples, as a result of the study by the present inventors, it has been clarified that the platelet concentration rate of PRP is related to the bone (or periodontal) tissue regeneration effect. Therefore, in order to obtain a higher regenerative effect, the platelet concentration rate ranges from about 150% to about 1500% (usually equivalent to about 240,000 / μL to about 6,150,000 / μL when converted to the average platelet count). It is preferable to use the PRP. More preferably, PRP having a platelet concentration rate in the range of about 300% to about 600% (usually corresponding to about 480,000 / μL to about 2,460,000 / μL in terms of average platelet count) is used.
A PRP having a desired platelet concentration can be obtained by appropriately adjusting the conditions of the centrifugal treatment in preparing the PRP. For example, the above-described two-stage centrifugation is performed, and the first centrifugation is performed under conditions of about 500 rpm to about 1500 rpm (for example, 1,100 rpm) for about 5 minutes to about 15 minutes (for example, about 5 minutes). By performing the second stage centrifugation under the conditions of about 2000 rpm to about 5000 rpm (eg, about 2,500 rpm) for about 5 minutes to about 15 minutes (eg, about 5 minutes), the platelet concentration rate is about 300% to about A PRP in the 600% range can be obtained. Although it is expected that the platelet concentration rate of the finally obtained PRP will fluctuate even if it is processed under the same conditions due to the difference in the starting material blood and the equipment used, those skilled in the art usually change the above conditions. The conditions for preparing PRP having a desired platelet concentration rate can be found by taking into account the correction of the conditions based on the platelet concentration rate of the obtained PRP.
The platelet concentration rate of PRP can be measured using a conventional method (for example, using commercially available Sysmex XE-2100 (Sysmex, Tokyo, Japan) as shown in the Examples).

  The platelet concentration rate of PRP used to construct the composition of the present invention is as described above, while the platelet concentration of the final composition (the composition of the present invention) is the platelet concentration rate of PRP, It varies depending on the use ratio of PRP and other components to be combined with it (that is, cells having osteogenic ability, etc.), for example, about 200,000 cells / μL to about 6,150,000 cells / μL, preferably about 480,000 cells / μL to about 2,460,000. Pieces / μL. By containing platelets at such a concentration, a good bone (or periodontal tissue) regeneration effect can be obtained. For example, if PRP with a platelet concentration rate in the range of about 300% to about 600% is used, the platelet concentration of the final composition should be adjusted to a range of about 480,000 / μL to about 2,460,000 / μL. Is possible.

Here, the degree of fluidity of the composition of the present invention at the time of use is not particularly limited, and may be a gel, slurry, paste, clay, high viscosity fluid, or the like. Preferably, it is a gel or paste. By setting it as a gel or paste, the composition for bone or periodontal tissue formation is excellent in plasticity. Therefore, it can be applied without previously forming the shape of the application portion. That is, application to the application unit can be easily performed. Moreover, it becomes the composition for bone or periodontal tissue formation with good fixing property in an application part.
In use, it is preferable that the fluidity is such that it can be injected using an injection container. By setting it as such fluidity | liquidity, the application to an application part becomes still easier.
In addition, it can be set as desired fluidity | liquidity according to an application part. For example, when injecting under the periosteum, it is preferable to have a more fluid state (low viscosity state).
The composition of the present invention only needs to have fluidity at least at the time of use, and may be powdery or solid before use. Therefore, it can be set as the composition of this invention with the frozen state. Moreover, it can also be set as the composition of this invention in the lyophilized | freeze-dried state. By making it frozen or lyophilized before use, long-term storage is possible, and handling before use is also facilitated. Furthermore, since a decrease in antigenicity can be expected by freezing treatment or freeze-drying treatment, safety is improved when using PRP of the same type other than PRP of the home.

In the present invention, “cells having the ability to form bone” refer to cells that can form bone tissue, and include osteoblasts, preosteoblasts, mesenchymal stem cells that have acquired the ability to differentiate into bone cells, and the like. . These cells may be used in any combination. Preferably, cells that have acquired the ability to differentiate into bone cells are used as the cells having the ability to form bone. Here, “obtained the ability to differentiate into bone cells” means a state in which an undifferentiated state is oriented to differentiate into bone cells.
As a mesenchymal stem cell that has acquired differentiation ability into bone cells, not only autologous cells but also allogeneic cells derived from the same species can be used. In particular, human mesenchymal stem cells can be used.
Mesenchymal stem cells that have acquired differentiation ability into bone cells (hereinafter referred to as “bone differentiation ability acquisition cells”) are cultured under conditions that induce differentiation of undifferentiated mesenchymal stem cells into bone cells. Can be prepared. For example, by differentiation of undifferentiated mesenchymal cells in a medium containing β-glycerophosphate, dexamethason, L-ascorbic acid, differentiation into bone cells Can be induced. Of course, culture conditions are not limited to this, and well-known conditions can be adopted as conditions for inducing differentiation into bone cells.

  Examples of undifferentiated mesenchymal stem cell sources include bone marrow, periosteum, dental pulp, and umbilical cord blood. These are collected according to a conventional method, and then undifferentiated mesenchymal cells are selected based on the presence or absence of adhesion. That is, undifferentiated mesenchymal stem cells can be obtained by selecting cells having adhesiveness among cells contained in bone marrow or the like.

  The composition of the present invention can be prepared by adding the extracellular matrix of the cells together with the bone system differentiation ability cells. This is because the extracellular matrix is expected to be a scaffold for bone differentiation-capable cells at the site to which the composition of the present invention is applied, and it is considered that the bone differentiation-capacity acquired cells are easily established at the application site. It is also a scaffold for bone cells existing around the application site, and high osteoinductive ability can be expected. Furthermore, factors such as BMP contained in the extracellular matrix have the ability to differentiate into bone system cells or bone cells around the application site, as well as the bone system differentiation ability acquiring cells themselves contained in the composition of the present invention. This is because it can be expected to promote the growth, proliferation and differentiation of stem cells. The extracellular matrix here is a matrix (matrix) that exists around cells that acquire bone differentiation ability.

Not only the extracellular matrix of autologous cells, but also the extracellular matrix of other types of autologous cells can be used. This is because BMP and the like contained in such an extracellular matrix are of the same type, and the same effect can be expected even when other cells are used. Thus, the ability to use not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells facilitates the preparation of the extracellular matrix of bone system differentiation-acquiring cells.
When an extracellular matrix is added, the bone system differentiation ability-added cells added at the same time may not be living cells. This means that it is not necessary to handle the bone system differentiation ability cells in the state of living cells, which can be said to be desirable from the viewpoint of handling. For example, after obtaining a bone system differentiation ability cell, a cell that has been lyophilized or the like can be prepared and used as the bone system differentiation capacity acquisition cell and its extracellular matrix.

In the composition of the present invention, the content of bone system differentiation-capable cells is preferably 1 × 10 ⁵ or more, more preferably 1 × 10 ⁶ to 1 × 10 ⁸ cells in 1 ml of the composition. Preferably, there are cells. This is because the bone formation can be effectively induced by setting the cell content.

  The composition of this invention is prepared by mixing said each component. As an example of the preparation method, a procedure for combining PRP and cells having osteogenic ability and forming a gel by the action of thrombin and calcium chloride (gelling material) will be briefly described. First, PRP and osteogenic cells are prepared and placed in one injection container. At this time, add a small amount of air. On the other hand, another injection container is prepared, and a mixed solution of calcium chloride solution and thrombin is put therein. Next, after these two injection containers are connected with a T-shaped connector, the plungers of both injection containers are alternately pushed (drawn) to mix the components in the two injection containers. The mixing operation is repeated until the desired fluidity (viscosity) is obtained.

  When the composition of the present invention is prepared in a frozen state or a lyophilized state, it is brought into a state having a desired fluidity at the time of use. In the case of a frozen state, the state before freezing is restored by thawing. At this time, physiological fluid etc. can be added and it can also adjust to desired fluidity | liquidity. On the other hand, in the lyophilized state, a solvent such as physiological saline is added to obtain a fluidity before lyophilization treatment or a desired fluidity.

  By encapsulating the composition of the present invention in an injection container, an injection for forming bone or periodontal tissue (hereinafter also referred to as “injection of the present invention”) 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 lyophilized to obtain the injection of the present invention. By using such an injection, handling becomes easier. That is, the desired effect can be expected by injecting the injection of the present invention transcutaneously or transmucosally without making an incision in the skin or mucous membrane as in the past. Such an injection is used after the bone or periodontal tissue-forming composition enclosed therein is made into a state having a desired fluidity as described above. The kind of injection container is not specifically limited, For example, a commercially available syringe can be used.

As described above, the composition of the present invention is characterized by the combination of platelet-rich plasma (PRP) and cells having bone forming ability, but the bone (or periodontal tissue) regeneration effect is retained. However, it does not prevent other components from being contained. However, in a preferred embodiment of the present invention, the composition is substantially composed of platelet-rich plasma (PRP) and only cells having bone forming ability (containing a component that promotes gelation when used).
The components that can be assumed to be additionally combined in the composition of the present invention are listed below.

(Inorganic bioabsorbable material)
The type of inorganic bioabsorbable material is not particularly limited, but β-tricalcium phosphate (“β-TCP), α-tricalcium phosphate (α-TCP), tetracalcium phosphate, octacalcium phosphate, and A material selected from the group consisting of amorphous calcium phosphate can be used, and these materials can be used alone or in combination of two or more selected arbitrarily. , Β-TCP, α-TCP, or a combination thereof in any proportion, more preferably β-TCP is used as the inorganic bioabsorbable material.
The inorganic bioabsorbable material can be obtained by a known method. Moreover, a commercially available inorganic bioabsorbable material can also be used. As β-TCP, for example, those manufactured by Olympus Optical Co., Ltd. can be used.

The inorganic bioabsorbable material is preferably in the form of a powder having a particle diameter such that the composition of the present invention becomes fluid when used.
The powdered inorganic bioabsorbable material can be prepared by crushing and pulverizing the inorganic bioabsorbable material processed to an appropriate size until a desired particle diameter is obtained. The average particle diameter of the inorganic bioabsorbable material is preferably 0.5 μm to 50 μm. More preferably, an inorganic bioabsorbable material having an average particle size of 0.5 μm to 10 μm is used. Even more preferably, an inorganic bioabsorbable material having an average particle diameter of 1 μm to 5 μm is used. 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 is preferably contained in an amount of 30% to 75% by weight based on the entire composition of the present invention.
In addition, the fluidity | liquidity of the composition of this invention can be adjusted with the particle diameter and content rate of an inorganic type bioabsorbable material, and desired fluidity | liquidity can be obtained by adjusting both suitably. Moreover, when adding the below-mentioned thickener, fluidity | liquidity can be adjusted also with the addition amount of a thickener.

(Gelling material)
For example, thrombin and calcium chloride can be added to form the composition of the present invention. By adding these, thrombin acts on fibrinogen in PRP to produce fibrin. And viscosity increases by the aggregation action of fibrin. The type of the gelling agent is not particularly limited, and those that act on the components in PRP to increase the viscosity as described above or those that exhibit a thickening effect by themselves can be appropriately selected and used.
In addition to the gelling material described above, a second gelling material that acts after application (after transplantation) to change the fluidity (viscosity) of the composition of the present invention can also be used in combination. With such a configuration, since it has appropriate fluidity at the time of use, it is easy to transplant, and after application, the viscosity increases to improve the fixability at the application site, and the bone or periodontal tissue Repair or regeneration can be performed effectively. Moreover, it is not necessary to mold into the shape of the application site in advance, and versatility is high.
As the gelling material, a material having high biocompatibility is preferably used, and collagen or fibrin glue or the like can be used in addition to the above examples. Various types of collagen can be selected and used, but it is preferable to employ one 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.).

(Thickener)
The fluidity of the composition of the present invention can be adjusted by adding a thickener. As the thickener, thickening polysaccharides such as sodium alginate, glycerin, petrolatum and the like can be used, but from the viewpoint of safety and / or bone forming ability, the biocompatibility is high and the bioabsorbable or biocompatible It is preferable to use a decomposable one. By adding glycerin or the like, the effect of preventing frost damage can be obtained.

(solvent)
The composition of the present invention may contain an aqueous solvent. As the aqueous solvent, sterilized water, physiological saline, a buffer solution such as a phosphate solution, or the like can be used.

(Other)
The composition of the present invention may contain a stabilizer, a preservative, a pH adjuster and the like in addition to the above components. Growth factors, particularly osteoinductive factors (BMP) can also be included.

<Materials and methods>
1. Dog Animal Model All animal experiments in this study were performed in strict accordance with protocols approved by the Animal Experiment Committee. After predetermined breeding, 4 adult hybrid dogs (average 2 years old) were treated under general anesthesia. The mandibular first premolar, premolar, and second and third premolars were extracted, and the healing period was set to February. A bone defect was formed on both sides of the lower jaw using a trephine bar with a diameter of 10 mm so as to be perpendicular to the lateral cortex. The following transplant materials, ie, PRP, PRP and canine MSCs (dMSCs), PCBM, and control (defects only) were transplanted into the bone defect thus formed, and bone formation was observed. Three bone defects were created in bone regeneration, and these four kinds of materials were randomly transplanted into three transplants so that there was no regional difference. PCBM was collected from the iliac crest.

2. MSC isolation and culture
dMSCs (canine mesenchymal stem cells) were punctured from canine iliac crest bone marrow and isolated according to a previously reported method (Non-patent Document 6). Briefly, basal medium, low glucose DMEM and growth supplement (50 mL of mesenchymal cell growth additive, 10 mL of 200 mM L-glutamine, and penicillin-streptomycin mixture containing 2.5 units of penicillin and 25 μg streptomycin (0.5 mL) was purchased from BioWhittaker (Walkersvill, MD, USA). Three additives for inducing osteogenesis, namely dexamethasone (Dex), β-glycerophosphate sodium (β-GP), and L-ascorbic acid diphosphate (AsAP) were added to Sigma (St. Louis, (MO, USA). Cells were cultured at 37 ° C. in a humidified atmosphere of 95% air and 5% CO ₂ . Control medium was inoculated with dMSCs at a concentration of 3.1 × 10 ³ cells / cm ² . Differentiated dMSCs were confirmed by alkaline phosphatase (ALP) staining with alkaline phosphatase using p-nitrophenyl phosphatase as a substrate. The dMSCs cultured in the control medium had low ALP activity, and according to the results after 6 days of treatment when cultured in a medium containing an osteogenic additive, the ALP activity increased dramatically over time. After culturing, dMSCs were trypsinized and used for transplantation.

3. Preparation of PRP, PRP gel, and injection of MSCs / PRP mixture Approximately 50 mL of whole blood was collected from the dog and placed in a centrifuge tube containing 10 mL of culture medium along with preservative-free heparin (250 U / mL). The blood was first centrifuged at 1100 rpm for 5 minutes using a standard laboratory centrifuge (Himac CT, Hitachi, Tokyo, Japan). Subsequently, yellow plasma (including buffy coat containing platelets and leukocytes) was cannulated to collect a monolayer intermediate layer. In order to pellet the platelets, a second centrifugation was performed at 2500 rpm for 5 minutes. And the plasma supernatant which is platelet poor plasma (PPP) and has a relatively small cell content was removed. The resulting platelet pellet, ie the buffy coat / plasma fraction (PRP), was suspended in the remaining 5 mL of plasma and used for platelet gel. Platelet counts in PRP and PPP were measured with Sysmex XE-2100 (Sysmex, Tokyo, Japan). As a result, the total platelet count was an average value of 295,000 (range 224,000 to 333,000). On the other hand, the platelet count of PRP averaged 1,293,400 (range 935,000 to 1,840,000). From these measurement results, it was confirmed that platelets could be separated, and the degree of concentration in PRP was found to be 438% (vs. whole blood platelet count (100%)). PRP was stored at room temperature in a conventional shaker until use. Powdered bovine thrombin (10,000 units) was dissolved in 10 mL of 10% calcium chloride solution in a separate sterile container. Next, 3.5 mL of PRP, dMSCs (1.0 × 10 ⁷ cells / mL), and 0.5 mL of air are aspirated into a 5 mL syringe, and 500 μL of thrombin-calcium chloride mixture is added into the second syringe (2.5 mL). Was aspirated. Here the cells were directly resuspended in PRP. The two syringes were connected by a three-way stopcock and mixed alternately in both syringes, so that air bubbles would flow between the two syringes. Due to the effect of thrombin, which acts on fibrin to form an insoluble gel, the contents appeared to exhibit a gel-like viscosity within 5-30 seconds. The resulting gel was injected into the bone defect using a 16 gauge needle attached to a 5 mL syringe. Each specimen was analyzed 2 weeks after injection (n = 6), 4 weeks (n = 6), and 8 weeks (n = 6). Lateral radiographs were taken under general anesthesia for radiological evaluation.

4). Histological and histomorphometric analysis After 2 weeks, 4 weeks and 8 weeks after transplantation, each transplanted part was cut out using a trephine bar (diameter 2 mm) for histological and histomorphometric analysis Provided. Specimens were fixed with 10% formalin buffer, decalcified (K-CX; Falma, Tokyo, Japan), and subjected to hematoxylin and eosin staining. These specimens were observed under an optical microscope, and the presence or absence of bone formation, including the author, was also analyzed blindly by pathologists.
The data of histomorphometric analysis was imaged with a microcomputer. Each image of the specimen in the transplanted part (diameter 2 mm) cut out with trephin bar was copied to a color reversal film, digitized as a 256 x 256 array of 8-bit density values, and transferred to a microcomputer for analysis (NIH -Image, version 1.61; National Institutes of Health) (Non-Patent Document 15). The regenerated area was defined as the area contained in the mandible cut with a trephine bar (diameter 10 mm). The amount of new bone in the regenerated area was measured with this computer-based image analysis system. It was calculated as the percentage of bone present and the normal bone site was subtracted from the measurement area.

5. Statistical analysis The mean value and standard deviation of each group were calculated for each measurement parameter. Differences in new bone among the control group, PRP group, dMSCs / PRP group, and PCBM group were analyzed by analysis of variance (ANOVA). Mann-Whitney evaluated the difference in new bone during each treatment period. Statistically significant when p value <0.05.

6). Relationship between platelet concentration rate of PRP and bone regeneration effect PRPs with different platelet concentration rates were prepared by changing the conditions of centrifugation (platelet concentration rate: 100-1500%). For each PRP, the bone regeneration effect when combined with dMSC was evaluated and compared by the same method as described above.

<Result>
1. Formation of Bone Defect Model in Canine Mandible FIG. 1 shows an experimental method for forming a 10 mm long defect in a dog's mandible in order to obtain an environment where bone does not regenerate naturally. The bone regeneration ability of the implant (implant) was evaluated by radiological, histological examination, and histomorphological analysis.

2. In vivo macroscopic, X-ray, and histological evaluation compared to PRP, dMSCs / PRP, and PCBM controls
PRP, dMSCs / PRP, and PCBM were transplanted into a 10 mm defect in the canine mandible. Macroscopically, it was at the original bone level in the dMSCs / PRP group and the PCBM group, but was not completely restored and regenerated in the PRP regeneration and control groups. The dMSCs / PRP scaffold disappeared almost completely without post-transplant infection (FIG. 2A, panels ac).
Bone regeneration and implants were evaluated by performing radiography and histological evaluation every two weeks. In the PRP group and the control group, bone regeneration slowly spread from the bottom of the defect. The defect filled with PCBM was radiolucent at 8 weeks after transplantation. This indicates that PCBM has been absorbed. On the other hand, good bone formation was observed in the defect filled with dMSCs / PRP, suggesting that bone formation occurred at almost the same rate as the resorption of the implant (FIG. 2B). Each region of the transplanted part and the control group (without transplantation) was collected after 2 weeks, 4 weeks, and 8 weeks, subjected to predetermined treatment and decalcification, and subjected to histological analysis. X-ray evaluation supported the results of histological analysis. In the control group and the PRP group, the continuity of the cortical marginal bone did not recover, the vascular and fibrous tissues invaded the defect, and almost no new bone formation was observed (FIGS. 3 and 4, panels AC). And DF). On the other hand, in the dMSCs / PRP group, new bone formation was observed after 2 weeks, and a lamellar structure was observed after 8 weeks, and abundant blood vessel formation was also confirmed (FIGS. 3 and 4, panel GI). ). This pattern reflects normal bone macrostructures, with well-differentiated bone marrow cavities and cortical bone compared to the PCBM group that showed cavities due to PCBM absorption (Figures 3 and 4, panel H and K).

3. Histomorphometric analysis The bone regeneration ability of all implants was evaluated by measuring the cortical bone and bone marrow bone surfaces by image analysis (Table 1). When PRP was added to the defect, no significant increase in the cortex and bone marrow bone surface was observed compared to the control group. In contrast, in the dMSCs / PRP group and PCBM group, the control group (p <0.001 after 2 weeks and 8 weeks, p <0.005 after 4 weeks, ANOVA) and the PRP group (after 2 weeks and 4 weeks p <0.05). , 8 weeks later (p <0.001, ANOVA), a significant increase in cortical and bone marrow bone surfaces was observed at all measurement points, confirming the results of X-ray and histological experiments. However, there was no significant difference in the new bone in the long term between the dMSCs / PRP group and the PCBM group.

4). Relationship between platelet concentration rate of PRP and bone regeneration effect Several PRPs with different platelet concentration rate were prepared. For each PRP, the bone regeneration effect when combined with dMSC was evaluated and compared. As a result, good bone regeneration was observed when the platelet concentration rate of PRP was in the range of 150% to 1500%. In particular, when the platelet concentration rate is in the range of 300% to 600%, the bone regeneration effect is very high.

<Discussion>
In the tissue engineering approach, attempts have been made to form new bone using MSCs seeded on a porous ceramic scaffold (Non-Patent Documents 7, 16, and 17). These attempts have led to suboptimal results due to the slow absorption rate of hydroxyapatite-based ceramics. In our previous study, we used a β-TCP block, which is a biodegradable material, with MSCs retained, and as a result, we found excellent bone-forming ability (Non-patent Document 8). However, these delivery materials do not have good plasticity, the implantation procedure is complicated and there are problems with the means of implantation. Optimally, these materials are required to have an appropriate rate of biodegradation and the ability to allow the growth of each cell. In this study, we used a combination of PRP and MSCs and found that it resulted in a progressive and complete resorption of the scaffold and formed relatively mature regenerated bone. According to our knowledge, when used in combination with MSCs, the biomaterial (PRP) has almost completely disappeared and replaced early with mature bone with the appropriate structure, which Bone regeneration was demonstrated. This result was obtained for the first time. In the control group, the defect was surrounded by soft tissue and did not heal. This confirmed that the size of the set defect is appropriate for evaluating the effect of the experiment. We have also found that the degree of healing varies significantly depending on the cell source. When the defect was filled with PRP alone, sufficient bone formation did not occur. By improving the culture method of pluripotent MSCs from bone marrow (Non-patent Document 18) and showing that it is possible to direct the differentiation of MSCs to the osteoblast lineage, It has been suggested that MSCs can be used. Certainly, MSCs can repeat cell division many times without losing bone forming ability (Non-patent Document 19). By proliferating culture of MSCs, it is possible to create a large number of cells capable of osteogenesis that could be used for clinical bone formation and repair (Non-patent Document 7). On the other hand, good results were obtained in tissue-engineered regenerative bone with dMSCs / PRP, suggesting that PRP had a positive effect on MSCs. The PRP scaffold is thought to promote the adhesion, proliferation and differentiation of MSCs and to exert bone forming ability. Since bone formation requires the supply of fully developed blood vessels (Non-Patent Document 20), it is considered that angiogenesis occurs in the transplanted scaffold. We have found that angiogenesis occurs well in the dMSCs / PRP group. Ideally, the scaffold needs to be absorbed at a rate that is balanced with new bone formation (a few weeks). Most hydroxyapatite, β-TCP ceramics, or sputum scaffolds (Non-patent Document 17) do not cause substantial degradation within a few weeks after implantation. On the other hand, in the experimental system of this study, it is probable that bone tissue formation was induced in response to dMSCs / PRP disappearance, and bone formation by autologous cells according to the surrounding environment was performed.
The average rate of angiogenesis in the rabbit ear is estimated to be 0.09 to 0.25 mm / day (Non-patent Document 21). Assuming comparable rates in dogs, blood vessels should reach the center of the implant within at least 20 days. Although many cell deaths may occur at the implant center due to lack of angiogenesis, the results obtained with tissue-engineered regenerative bone in this study show good cell viability and MSCs in osteogenesis. Direct involvement is suggested. Therefore, it is considered that PRP promoted angiogenesis.

Proliferation of undifferentiated mesenchymal stem cells, differentiation into osteoprogenitor cells (osteoprogenitor, preosteoblast), maturation of osteoblasts, matrix (type I collagen) formation, and final Osteogenesis occurs as a result of a complex cascade event with typical calcification (22, 23). The first event would be the induction of osteoblast chemotaxis. Owen and Friedenstein show that bone marrow and peripheral progenitor cells produce bone and cartilage in numerous in vivo and in vitro studies and show that the differentiation process is strongly influenced by many cytokines. (Non-Patent Document 5). In the system of this study, the use of dMSCs / PRP has become a condition for achieving faster and more effective bone regeneration. PRP is an autologous platelet-derived growth factor (PDGF) source, and includes transforming growth factor β (TGF-β) and the like. In addition, the PRP gel that is an agglomerate has good operability. However, it is necessary to apply it quickly considering the maintenance of growth factor activity. The survival time of platelets in the wound and the period during which the direct effect of the growth factor is obtained is within 5 days (Non-patent Document 24). In addition to these growth factors, other proteins in platelets (Non-Patent Document 25) will work with cytokines released from other cell sources to regulate hemostasis. The results of this study suggest that enhanced growth factor concentrations by application of PRP in wounds improved soft tissue repair and bone regeneration.
Khouri et al. Have succeeded in experimentally forming in vivo various self-shaped and well-vascularized bones of various desired shapes by tissue transformation (tissue transformation). This tissue transformation is a transformation of mesenchymal tissues such as muscle, cartilage, and bone, and is induced by the osteoinductive factor osteogenin, which is the same as BMP-3, and its parent substance, DMP (demineralized bone matrix). It is induced (Non-patent Document 26). Despite the solid concept and the validity of experimental data, this approach has not been widely used. The reason is that it is difficult to steadily supply DBM that is licensed for clinical use, and DMB must be prepared from cadaveric bone, and is not approved in Japan. More importantly, since the inductivity varies depending on the DBM used, most surgeons cannot attempt to use it even for distal muscle flap implantation and tissue shaping (27). . This method also involves invasion of other sites. Furthermore, it is difficult to purify osteogenin easily and without toxicity or immunogenicity problems. On the other hand, our method involves autologous bone regeneration by tissue engineering, which is non-toxic, non-immunogenic, can be performed with minimal invasiveness and provides excellent plasticity.

In summary, our study shows that dMSCs / PRP implants, like autologous bone grafts, can induce true bone regeneration, which is a complete biomaterial in bone defects of clinically significant volume. It was shown to be accompanied by loss and formation of bone / cartilage tissue by PRP. In addition, since PRP is a self-preparation and can be prepared at the time of surgery, there is no risk of infection and immune reaction associated with the use of other homes or heterogeneous preparations. There is no fear. In addition, the dMSCs / PRP mixture can be applied by injection, coagulating in the host and being replaced with bone over time, making it a very promising method in the future for orofo-maxillofacial area and reconstruction.
The method presented in this study is an important step towards customized autologous bone grafting. Theoretically, autologous MSCs can be obtained by minimally invasive biopsy, followed by inducing MSCs into cells that differentiate in vitro as osteoblasts and then re-implanted in a controlled manner. Direct contouring, reconstruction, and bone regeneration for periodontal disease, dental implants. Experimental data obtained for dMSCs / PRP mixtures showed that MSCs migrate efficiently into and through PRP. PRP promotes the growth of MSCs without deforming the cell structure and acts as a suitable delivery agent. PRP would be useful for repairing or reconstructing bone defects in the clinical field as a highly suitable vehicle for delivery of cells by injection.

  The composition for forming bone or periodontal tissue of the present invention can be applied to various fields in which repair or regeneration of bone tissue or periodontal tissue is required. For example, the present invention can be applied to bone augmentation in the case where an artificial tooth root is planted on a jaw crest where a high degree of bone resorption is recognized. In addition, the present invention can be applied to bone tissue regeneration in bone defects caused by trauma and various bone diseases, and bone reinforcement or supplementation. Further, the present invention can be applied to the regeneration of alveolar bone and periodontal tissue in the alveolar bone defect due to periodontal disease and the like. As an application method, a gel-like or paste-like composition is filled into the application site, injected, or applied.

  The composition for forming bone or periodontal tissue of the present invention contains a number of growth factors having the ability to promote the proliferation or differentiation of cells of the osteogenic system. Therefore, in an application part (transplantation part), the cell of a bone formation type | system | group can be efficiently proliferated or differentiated, and formation of a bone tissue or periodontal tissue can be accelerated | stimulated. By using autologous PRP, it is considered that the regeneration of bone tissue or periodontal tissue is improved in both quality and quantity by the above-mentioned growth factor which is non-toxic and immune inactive. In addition, since it is fluid, such as a gel, it can be easily inserted into the application part using an injection needle or the like (it can also be applied without opening the wound), and the shape of the bone defect part It does not require pre-molding together, and its versatility is high. Thus, by using the composition for forming bone or periodontal tissue of the present invention, it is not necessary to collect autologous bone and use it for transplantation, and it is possible to easily increase the growth of bone or periodontal tissue. .

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

An experimental protocol is shown. (A) Macroscopic findings of bone regeneration: (a) Experimental design in the mandible of the dog, produced with a trephine bar with a diameter of 10 mm. (b) Transplant material in the bone defect. (c) Bone regeneration in dMSCs / PRP group, PCBM group and control group (8 weeks after transplantation). The dMSCs / PRP group and PCBM group were at the original bone level but were not completely regenerated in the PRP or control group. (B) Evaluation by X-ray: (a) X-ray image after transplantation. (b) X-ray image 2 weeks after transplantation. Note that there is no bone formation in the defect of the control group. Transplanted PCBM is observed in the PCBM group, and bone formation is observed in the dMSCs / PRP group. (c) X-ray image 4 weeks after transplantation. In the dMSCs / PRP group and PCBM group, unlike the control group, bone formation is observed in the defect. (d) X-ray image 8 weeks after transplantation. The defect filled with PCBM is radiolucent after 8 weeks and shows PCBM absorption. In contrast, implants with dMSCs / PRP implants showed good bone formation. (A-L) Histological evaluation after 2, 4, and 8 weeks of control group, PRP group, PCBM group, and dMSCs / PRP group: low magnification. Representative implant cuts for each group. Stained with hematoxylin and eosin. Original magnification: (A-L) x 40. (A) 2 weeks after control group; (B) 4 weeks after control group; (C) 8 weeks after control group; (D) 2 weeks after PRP group; (E) 4 weeks after PRP group; (F) 8 weeks after PRP group; (G) 2 weeks after dMSCs / PRP group; (H) 4 weeks after dMSCs / PRP group; (I) 8 weeks after dMSCs / PRP group; (J) PCBM 2 weeks after group; (K) 4 weeks after PCBM group; (L) 8 weeks after PCBM group. (A-L) Histological evaluation after 2, 4, and 8 weeks of control group, PRP group, PCBM group, and dMSCs / PRP group: low magnification. Representative implant cuts for each group. Stained with hematoxylin and eosin. Original magnification: (A-L) x 40. (A) 2 weeks after control group; (B) 4 weeks after control group; (C) 8 weeks after control group; (D) 2 weeks after PRP group; (E) 4 weeks after PRP group; (F) 8 weeks after PRP group; (G) 2 weeks after dMSCs / PRP group; (H) 4 weeks after dMSCs / PRP group; (I) 8 weeks after dMSCs / PRP group; (J) PCBM 2 weeks after group; (K) 4 weeks after PCBM group; (L) 8 weeks after PCBM group. Histological evaluation after 2, 4, and 8 weeks of the control group, PRP group, PCBM group, and dMSCs / PRP group: high magnification. Representative implant cuts for each group. Stained with hematoxylin and eosin. Original magnification: (A-L) × 200. (A) 2 weeks after control group; (B) 4 weeks after control group; (C) 8 weeks after control group; (D) 2 weeks after PRP group; (E) 4 weeks after PRP group; (F) 8 weeks after PRP group; (G) 2 weeks after dMSCs / PRP group; (H) 4 weeks after dMSCs / PRP group (active angiogenesis is observed); (I) dMSCs / PRP group 8 weeks later (stratified bone is observed); (J) 2 weeks after the PCBM group (arrow, transplanted PCBM); (K) 4 weeks after the PCBM group (cavity is seen, due to resorption of transplanted PCBM) ; (L) 8 weeks after PCBM group. It is the table | surface which put together the histological analysis result. The bone regeneration ability of all implants was evaluated by measuring the cortical and medullary bone surfaces with image analysis. Significance: * p <0.005, ** p <0.001

Claims (8)

  A composition for forming bone or periodontal tissue, comprising platelet-rich plasma (PRP) having a platelet concentration rate in the range of about 150% to about 1500% and cells having osteogenic potential.   2. The composition of claim 1, wherein the platelet concentration is in the range of about 300% to about 600%.   A composition for bone or periodontal tissue formation comprising platelet rich plasma (PRP) and cells capable of osteogenesis, wherein the platelet concentration is in the range of about 200,000 / μL to about 6,150,000 / μL.   A composition for forming bone or periodontal tissue, comprising platelet-rich plasma (PRP) and cells capable of forming bone.   It has the fluidity | liquidity which can be inject | poured using an injection | pouring container at the time of use, The composition in any one of Claims 1-4 characterized by the above-mentioned.   The composition according to any one of claims 1 to 5, wherein the cells are mesenchymal stem cells that have been induced to differentiate into bone cells. The composition according to any one of claims 1 to 6, which is a composition applied to a dental implant, periodontal disease, cyst, tooth extraction socket, bone extension, or cleft bone graft site.   The injection for bone or periodontal tissue formation formed by enclosing the composition in any one of Claims 1-7 in an injection | pouring container.