JP2008297220A - Preventive and therapeutic agents for bone diseases with fractures and bone loss - Google Patents

Abstract

Provided is a method for notably enhancing differentiation induction from mesenchymal cells other than mesenchymal stem cells and osteoblasts into osteoblasts, but also significantly promoting osteoblast differentiation and maturation. Furthermore, the present invention provides a prophylactic and therapeutic agent for bone diseases accompanied by fractures and bone loss, and particularly a drug applicable to a stent device for transplantation and artificial bone prosthetic materials necessary for regenerative treatment in bone diseases such as fracture treatment and bone loss.
A method of promoting differentiation from mesenchymal cells to osteoblasts by using platelet-rich plasma and osteogenesis-inducing protein in combination; also promoting differentiation and maturation of osteoblast precursor cells and / or osteoblasts A preventive and therapeutic agent for bone diseases accompanied by fracture and bone loss, wherein the therapeutic agent comprises a combination of platelet-rich plasma and osteogenesis-inducing protein; mesenchymal system An osteoblast for the prevention and treatment of bone diseases accompanied by fracture and bone loss, obtained by culturing cells in the presence of platelet-rich plasma and osteogenesis-inducing protein.
[Selection figure] None

Description

  The present invention not only strongly enhances (1) transdifferentiation induction from mesenchymal cells other than osteoblasts to osteoblasts, but also (2) osteoblast precursor cells and osteoblasts. The present invention relates to the prevention and treatment of bone diseases in which differentiation and maturation are significantly promoted and bone formation and bone regeneration are required due to fracture and bone loss.

  Bone is a hard tissue, but it is a dynamic tissue that remodels by repetitive bone resorption by osteoclasts and bone formation by osteoblasts. Bone tissue is kept constant due to the balance between bone formation and bone resorption, but bone diseases with bone loss, such as osteoporosis, lose this balance and relatively reduce bone formation, It is known to develop with increased absorption. One of the major social problems in an aging society is the increase in patients with osteoporosis and the increase in bedridden elderly people due to fractures, etc., and development of therapeutic drugs is desired. Many bone diseases, such as osteoporosis, joint bone destruction in rheumatoid arthritis, and reduced alveolar bone mass due to periodontal disease, occur when bone resorption exceeds bone formation. Therefore, agents that suppress bone resorption, such as bisphosphonate compounds, have been used as first-line drugs as therapeutic agents for bone diseases associated with bone loss.

  The present inventors have already reported that osteoclastogenesis inhibitory factor (osteoprotegerin, OCIF / OPG) (Tsuda E., Higashio K., et al .: Biochem) as a key factor involved in osteoclast formation and regulation. Biophys. Res. Commun. 234: 137-142, 1997; Yasuda H., Higashio K., et al .: Endocrinology 139: 1329-1337, 1998) and osteoclast differentiation factor / receptor- activated NF-κB ligand (ODF / RANKL) (Yasuda H., Higashio K., et al .: Proc. Natl. Acad. Sci. USA 95: 3597-35602, 1998) Elucidated the molecular mechanism of. It was possible to develop an ideal and ultimate bone resorption inhibitor based on this molecular mechanism. A fully humanized antibody (AMG162) against RANKL, currently under clinical development in the United States, has been reported to have excellent clinical effects in postmenopausal women by significantly suppressing bone resorption by subcutaneous administration of 60 mg each twice a year. (Takahashi N. and Ozawa H .: Clin. Calcium 15: 43-48, 2005). As described above, a method for treating a bone metabolic disease with a bone resorption inhibitor is being almost established.

  However, bone loss in bone diseases that develop due to increased bone resorption has a major problem that even if treated with a bone resorption inhibitor, it cannot be restored to the original bone mass, and actively promotes bone formation. Development of therapeutic agents that can be used is desired. There are female hormones (estrogens, E2) as osteogenesis promoters and parathyroid hormone (PTH) by intermittent administration recently approved by the US FDA, both of which have been pointed to the risk of carcinogenesis. In the latter, the administration period is limited to 2 years. As described above, all of these drugs are concerned about carcinogenesis, and development of a highly safe osteogenesis promoter is desired.

  With the progress of an aging society, the need for bone regeneration treatment associated with osteoporosis, cartilage disorders, alveolar bone defects and the like is increasing. Currently, bone regeneration treatment methods include autologous bone transplantation, artificial bone grafting material transplantation, and heterogeneous bone transplantation. Among them, autologous bone transplantation is considered the gold standard (Boyapati.L. And Wang HL .: Implant Dentistry 15: 160 -168, 2006). However, this law includes various problems such as enormous burdens on patients, limited supply sources, long treatment periods, unhealthy postoperative conditions, and high treatment costs. is doing.

  In particular, the healing of fractures and the extension of the regenerative healing period of bone defect sites are major medical problems that remain unsolved. Many clinicians and researchers have studied various methods including the addition of growth-promoting factors to shorten this healing period. Among them, platelet rich plasma (PRP) prepared from autologous blood as a method for shortening the healing period, enhancing wound healing and promoting osteogenesis Transplantation was proposed (Max RE., Et al .: Oral Med. Oral Phathol. Oral Radiol. Endod. 85: 638-646, 1998; Lozada JL., Et al .: J. Oral Implantol. 27: 38- 42, 2001). PRP contains platelets concentrated at high concentrations in a small amount of plasma, and is characterized by high concentrations of several growth factors and adhesive glycoproteins. Specifically, PRP is an artificial bone grafting material mainly composed of calcium phosphate, represented by β-TCP (β-tricalcium phosphate), because it is easy to obtain and can be prepared at low cost. Replacement transplantation using PRP and PRP has been proposed. In fact, PRP prepared from autologous blood is being clinically applied to β-TCP, an artificial bone prosthesis, for the regeneration of alveolar bone prior to implants in the dental field.

  According to a recent review (Boyapati. L. and Wang HL .: Implant Dentistry 15: 160-168, 2006), the rationale for the use of PRP in the regeneration of bone defect sites is the vessel to the transplanted tissue (substrate) Includes accelerated newborn, improved soft tissue healing, postoperative health and enhanced bone regeneration. In addition, the action of these PRPs is contained in α-granule of platelets abundant in PRP, and is released by activation platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), TGF-β It is thought to be due to growth factors such as platelet-derived angiogenic factor (PDAF) and vascular endothelial growth factor (VEGF). PDGF promotes DNA and protein synthesis in bone tissue (Witfang J., et al .: Clin. Oral Implant Res. 14: 213-218, 2003), and promotes proliferation of mesenchymal cells (Roldan JC., Et al. : Bone 34: 80-90, 2004), known to have angiogenic effects on vascular endothelial cells, collagen and matrix formation. IGF promotes osteoblast proliferation and matrix synthesis, enhances expression of bone matrix proteins such as osteocalcin, and TGF-β promotes angiogenesis and enhances bone formation (Landesberg R., et al. : J. Oral Maxillofac. Surg. 58: 297-300, 2000; Ripamonti U., et al .: J. Bone Miner. Res. 12: 1584-1595, 1997; Fujimoto R., et al .: J. Bone Miner. Metab. 17: 11-17, 1999), promotion of cell matrix synthesis, migration effect on osteoblasts, and promotion of bone formation by osteoclast inhibition are known. PDAF has the effect of promoting the proliferation of vascular endothelial cells and promoting angiogenesis, and VEGF is known as a powerful angiogenic factor.

  Clinical application of PRP to the treatment of human periodontal defects (alveolar bone defects) (Hanna R., et al .: J Periodontol. 75: 1668-1677, 2004; Okuda K., et al .: J. Periodontol. 76 : 890-898, 2005;) and reports on the use of PRP for regeneration by the GBR (guided bone regeneration) method of alveolar bone whose bone mass has decreased due to periodontal disease (Kim SG., Et al .: Int. J. Oral Maxillofac. Implant 17: 86-94, 2002). There are also many reports on bone regeneration by implanting PRP into bone defect voids. However, there are currently many conflicting reports about the effects of PRP on bone formation or bone regeneration. For example, reports claiming efficacy in animal models (Furst G., et al .: Clin. Oral Implants Res. 14: 500-508, 2003; Ohya M., et al .: Clin. Oral Implants Res. 16: 622-629, 2005), but invalid (Roldan JC., Et al .: Clin. Oral Implants Res. 15: 716-723, 2004; Grageda E., et al .: J. Oral Implantol. 31: 2-17, 2005; Butterfield KJ., Et al .: J. Oral Maxillofac. Surg. 63: 370-376, 2005). In addition, the effect of PRP application in human implants is similar, and the effect of PRP on bone mass increase at bone defect sites is positive (Wiltfang J., et al .: Clin. Oral Implants Res. 14: 213-218, 2003; Graziani F., et al .: Implant Dent. 14: 63-69, 2005; Phillippart P., et al .: Int. J. Oral Maxillofac. Implants 20: 274- 281, 2005) and disabled (Froum SJ., Et al .: Int. J. Periodontics Restorative Dent. 22: 45-53, 2002; Raghoebar GM., Et al .: Clin. Oral Implants Res. 16: 349-356 , 2005) is actually present.

  Thus, it cannot be said that the effect of PRP on bone formation or bone regeneration has been well documented scientifically. In fact, a recent review of the role of PRP on bone defect regeneration enhancement (bone regeneration and wound healing) also recommends the use of PRP in bone regeneration enhancement treatment due to limited scientific evidence. (Boyapati. L. and Wang HL .: Implant Dentistry 15: 160-168, 2006).

  On the other hand, BMP (bone morphogenetic protein) is well known as an osteogenesis inducing factor that promotes the differentiation and maturation of osteoblasts responsible for bone formation. BMP was found approximately 40 years ago as a factor that induces bone formation in bone tissue (Urist MR .: Science 150: 893-899, 1965), and its cDNA was cloned by Wozney et al. (Wozney JM al .: Science 242: 1524-1534, 1988). Many energetic studies have identified nearly 20 BMP members, of which BMP2, BMP4, BMP6, BMP7 and BMP-9 are particularly involved in bone metabolism and bone formation. (Cheng H., et al .: J. Bone and Joint Surgery (American) 85: 1544-1552, 2003).

  BMP not only differentiates / maturates osteoblast progenitor cells and osteoblasts, but also differentiates mesenchymal stem cells and mesenchymal cells other than osteoblasts into osteoblasts (transdifferentiation) Is also an essential factor and is involved in osteogenesis through osteoblast mobilization, differentiation and maturation promotion.

  However, about 40 years have passed since BMP was discovered, and until now it has been developed as a treatment for bone diseases by promoting bone formation, but it is still on the market as a treatment for bone diseases including osteoporosis. Not reached. However, BMP2 and BMP7 (OP-1) are clinically applied to transplanted stent devices for fracture treatment in the orthopedic region in the West. As described above, there are autologous bone transplantation, artificial bone grafting material transplantation, and heterogeneous bone transplantation as bone regeneration treatment methods, and autologous bone transplantation is regarded as a gold standard. However, this method imposes a great burden on patients, and in particular, the prolonged treatment of fractures and bone defect regeneration treatments is regarded as a major medical problem that remains unsolved. BMP2 or BMP7 (OP-1) applied fracture healing stent device is superior to cold standard autologous bone grafts in clinical trials, although it is superior to BMP supplementation in the case of open fracture patients. There is no difference in the therapeutic effect between patients with closed fractures and spinal junctions, and no significant shortening of the treatment period (surgery / hospitalization period) has been observed in any patient. (Bone Morphogenic Protein for the Treatment of Long Bone Fractures and for Use In Spinal Fusion Procedures. Department of Labor and Industries Office of the Medical Director September 29, 2003)

  As described above, BMP has been developed as a therapeutic agent for bone diseases such as osteoporosis, but one of the major obstacles to the development of therapeutic agents for bone diseases is in vivo drug administration. The amount is very large, and development is considered difficult from the viewpoint of cost. In addition, even in the application of BMP in the implanted stent device for local fracture treatment that is currently in practical use, the dose is still several mg / device for local administration, compared to the gold standard autologous bone graft In some cases, the effect is only seen, and no significant shortening of the treatment period is observed. This is the reason why the development of a superior stent device for transplantation and artificial bone filling material is desired.

  The present invention not only enhances differentiation induction from mesenchymal cells other than mesenchymal stem cells and osteoblasts into osteoblasts (mobilization of osteoblasts) but also significantly promotes osteoblast differentiation and maturation. It is an object of the present invention to provide a method. The present invention further provides a preventive and therapeutic agent for bone diseases associated with fractures and bone loss, and in particular, a stent device for transplantation and artificial bone filling material necessary for regenerative treatment in bone diseases such as fracture treatment and bone loss (bone defect). The purpose is to provide a drug applicable to the drug.

  The treatment of bone diseases involving fractures, bone regeneration and bone loss requires the active promotion of bone formation. (1) Mesenchymal systems other than mesenchymal stem cells and osteoblasts In addition to enhancing the induction of transdifferentiation into osteoblasts responsible for bone formation from cells (mobilization of osteoblasts), (2) promotion of osteoblast differentiation and maturation is necessary.

  As a result of intensive studies on factors that satisfy these two conditions at the same time, the present inventors have used these in combination with platelet rich plasma (PRP) and bone morphogenetic protein (BMP). The present invention was completed by discovering that the above two objects could be achieved simultaneously.

That is, the present invention provides the following.
(1) A method of promoting differentiation from mesenchymal cells to osteoblasts by using platelet rich plasma (PRP) and bone morphogenetic protein (BMP) in combination.
(2) A method of promoting differentiation / maturation of osteoblast precursor cells and / or osteoblasts by using platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP) in combination.
(3) Use of platelet rich plasma (PRP) and bone morphogenetic protein (BMP) in combination promotes differentiation from mesenchymal cells to osteoblasts, and then A method of promoting cell differentiation / maturation.
(4) A preventive and therapeutic agent for bone diseases associated with fractures and bone loss, wherein the therapeutic agent comprises a combination of platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP). Therapeutic agent.
(5) The platelet-rich plasma (PRP) -derived active ingredient and bone formation-inducing protein (BMP) are administered together with a carrier selected from the group consisting of a collagen sponge and an artificial bone prosthetic material mainly composed of calcium phosphate, 4) The preventive and therapeutic agent for the bone disease accompanied by the fracture and bone loss described in the above.
(6) Osteoblasts for prevention and treatment of bone diseases with fractures and bone loss, obtained by culturing mesenchymal cells in the presence of platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP) .
(7) A prophylactic and therapeutic agent for bone diseases accompanied by fracture and bone loss, comprising the osteoblast according to (6).

  The platelet-rich plasma (PRP) of the present invention can be prepared from fresh blood such as humans, cows and pigs, but is prepared from autologous blood to avoid antigenic and infectious diseases. PRP is most preferred. In that case, it is preferable to prepare from fresh blood of a healthy person having a normal platelet count (150,000 to 400,000 / μL). For the preparation of PRP, the methods adopted in many prior literatures can be used. However, in order to standardize the preparation of PRP and to compare the in vivo efficacy of PRP with each other, two recently approved by the US FDA Harvest SmartPrep Platelet Concentrate System (Harvest Technologies, MA) or Platelet Concentration Collection System (3i Implant Innovations, FL), which is a PRP collection system (Boyapati L. and Wang HL .: Implant Dentistry 15: 160-167, 2006) It is preferable to use it.

  The PRP prepared by the above method may be used as it is, or a fibrin clot formed in advance by platelet activation treatment before adding PRP to the culture solution, and then centrifuged to cleave the fibrin clot. It is also possible to prepare activated PRP from which is removed and to use it. The activated PRP prepared in Examples described later corresponds to PRP in which platelets in the original plasma are concentrated about 10 times.

  As the bone formation-inducing protein of the present invention, various known bone morphogenetic proteins (BMP) can be used. Specific examples include BMP2, BMP4, BMP6, BMP7 and BMP9, which are known to be deeply involved in bone metabolism and bone formation. In fact, as shown in Examples 1 to 5, the combination with PRP Many of these BMPs (BMP2, BMP4, BMP6 and BMP7) are prominent in (1) inducing differentiation from mesenchymal cells to osteoblasts, and (2) promoting differentiation and maturation of osteoblast precursor cells and osteoblasts. To be enhanced. In addition to each of the above BMPs, as long as the activity is maintained, a part of the amino acids may be modified by deletion, substitution, addition or the like. The origin is not particularly limited, but human-derived BMP is preferred.

  Mesenchymal stem cells are undifferentiated cells that can differentiate into chondrocytes, muscles, osteoblasts, bone marrow stromal cells, adipocytes, and the like. In the present invention, PRP strongly enhances differentiation induction from mesenchymal stem cells by BMP and mesenchymal cells such as chondrocytes, muscles, bone marrow stromal cells and adipocytes into osteoblasts. Promotes differentiation and maturation of osteoblast precursor cells and osteoblasts by BMP. Examples of mesenchymal stem cells include undifferentiated mesenchymal stem cells that can differentiate into mesenchymal cells such as chondrocytes, muscles, osteoblasts, bone marrow stromal cells, and adipocytes. Lineage stem cells. Examples of mesenchymal cells other than osteoblasts include normal human preadipocytes (Cryo HPRAD-Visc and Cryo HPRAD-SQ, Sanko Junyaku Co., Ltd.), normal human aortic vascular smooth muscle cells ( Cryo AOSMC, Sanko Junyaku Co., Ltd.), myoblast cell line as cell line, C2C12 (Katagiri T., et al .: J. Cell Biology 127: 1755-1766, 2004), adipocyte progenitor cell line, MC3T3- L1 and chondrocyte progenitor cell lines, such as ATDC-5 (Nakamura K., et al .: Exp. Cell Res. 250: 351-363, 1999), and osteoblast progenitor cell lines such as ST2 and MC3T3-E1 Including, but not limited to.

When differentiating mesenchymal cells in vitro, the mesenchymal cells are suspended in a medium such as DMEM or αMEM containing fetal bovine serum (FBS) at an appropriate concentration (eg, 1-15%), and 80-90% After culturing until confluent, add BMP and PRP. Specifically, the minimum concentration of BMP that can normally induce alkaline phosphatase (ALP) activity alone (because the specific activity differs depending on the type of BMP, the host cell for production, and the degree of purification, it is difficult to specify the concentration of each BMP. For example, in each recombinant BMP preparation used in the present invention, BMP2 (host: E. coli) 300 ng / ml, BMP4 20-25 ng / ml, BMP6 100-150 ng / ml, BMP7 200 ng / ml The above, and activated PRP (corresponding to about 10 times the concentration of platelets compared to the original plasma) is replaced with 1-33%, particularly preferably 4-17% of the same medium, 37 ° C., Cultivate in the presence of 5% CO ₂ for 3-4 days to differentiate into osteoblasts.When osteoblasts are differentiated and matured, BMP and PRP may be newly added, but the culture is continued as it is. Usually, the osteoblasts can be differentiated and matured by culturing for 3 to 4 days, using a combination of PRP and BMP. Differentiation and maturation of differentiated preliminary bone osteoblasts from osteoblast precursor cells into osteoblasts also can be carried out in the same manner as described above.

  In the present invention, the osteoblasts thus obtained can be used for the prevention and treatment of bone diseases associated with fractures and bone loss. Specifically, osteoblasts obtained by the method of the present invention are transplanted to an affected area such as a fracture or a bone defect site (for example, alveolar bone regeneration) together with an artificial bone filling material, a stent device for transplantation, etc. And bone diseases associated with bone loss can be prevented and treated.

  In order to actively promote osteogenesis, (1) enhanced differentiation induction from mesenchymal stem cells and mesenchymal cells to osteoblasts (recruitment of osteoblasts) and (2) osteoblasts Promotion of differentiation and maturity is essential. As mentioned above, osteoblast precursor cells and osteoblasts are sensitive to BMP and express alkaline phosphatase in small amounts of BMP (<10 ng / ml) to differentiate and mature, but from undifferentiated mesenchymal cells The process of inducing differentiation into osteoblasts is extremely insensitive to BMP, so it is known that mobilization of osteoblasts does not proceed easily even with large amounts of BMP (> 100 ng / ml) .

  However, the combined use of PRP and BMP according to the present invention strongly enhances differentiation induction from mesenchymal cells to osteoblasts by BMP, and further differentiates osteoblast precursor cells into osteoblasts and osteoblasts. Remarkably enhances differentiation and maturation.

  The present invention can also be used for regenerative treatment (for example, alveolar bone regeneration) of a bone defect site due to fracture or bone loss in vivo. For example, by mixing PRP prepared from autologous blood or activated PRP and BMP, and mixing them in collagen sponge or artificial bone filling material, both BMP and PRP-derived active ingredients are adsorbed and retained on these carriers. It can be carried on a carrier. Carriers capable of adsorbing and retaining both BMP and PRP-derived active ingredients are selected from the group consisting of collagen sponges and artificial bone filling materials based on calcium phosphate such as β-TCP and porous hydroxyapatite (HA) .

The PRP of the present invention or the combined agent of activated PRP and BMP is preferably administered locally, and in this case, the dose is 0.1 to 100 mg / dose site / person in the case of fracture treatment. Preferably in the range of 0.5-10 mg / site / person, and PRP is converted to activated PRP (equivalent to approximately 10 times the concentration of the original plasma platelets), and the volume of the artificial bone graft material to be transplanted (Cm ³ ) is selected in a range corresponding to about 1 to 33%, more preferably about 2 to 25%. However, the above addition amount range is applied when the active ingredient derived from BMP4 and PRP is efficiently adsorbed and retained (90% or more) like the collagen sponge used in Example 5. Since the adsorption / retention rate of the active ingredient derived from BMP and PRP differs depending on the difference, the addition amount range needs to be corrected by their adsorption / retention rate. Thus, the effective addition amount of each component derived from BMP and PRP can be effectively mixed with one selected from the group consisting of collagen sponge and artificial bone filling material mainly composed of calcium phosphate, or it can be effectively infused and transplanted. Can prevent and treat severe fractures.

Also, in bone regeneration treatment in a relatively small area such as regenerative treatment of alveolar bone, BMP is in the range of 0.1 to 1000 μg / administration site, and activated PRP is the transplantation volume (cm ³ ) of the artificial bone filling material. Is selected in a range corresponding to about 1 to 33%, more preferably about 2 to 25%. However, since the above-mentioned addition range is when the adsorption / retention rate is very high (90% or more), the adsorption / retention rate differs depending on the material and structure, etc., so the above-mentioned addition range needs to be corrected accordingly There is.

The effect of the present invention was confirmed as follows.
As described in Example 1, plasma was prepared by first-stage centrifugation of fresh blood obtained by collecting blood from healthy humans using an anticoagulant (3.8% sodium citrate). Plasma is obtained in about half of the blood volume used, and platelets are concentrated and precipitated at the bottom of the centrifuge tube by high-speed centrifugation in the second stage. The concentrated platelets were resuspended to 1/100 volume of the original plasma with platelet poor plasma (PPP) in the supernatant (platelets were concentrated to about 100 times the original plasma). As shown in Example 1, the present inventors have used a myoblast cell line, C2C12 (Katagiri T., et al .: as a mesenchymal cell that is already known to differentiate into osteoblasts by BMP2 stimulation. J. Cell Biology 127: 1755-1766, 2004) was used. Add 0-5 μL of human PRP to each well (culture medium 150 μL / well) of the 96-well plate in which C2C12 cells were cultured, and add 5-10 μL of human PPP so that the amount of human plasma added to each well is 10 μL. Adjusted. When the plate thus prepared was cultured, PRP was coagulated and activated in the culture medium, but the addition of PRP alone did not show any ALP-inducing activity of C2C12 cells, which are mesenchymal cells. Therefore, as a result of intensive studies on the combined effect of PRP and other factors, the present inventors have found that PRP strongly enhances differentiation induction from mesenchymal cells to osteoblasts when used in combination with BMP. I found it. As shown in Example 1, an unexpected effect was observed that PRP strongly enhanced ALP-inducing activity by BMP4 in the presence of a low concentration of BMP4 (20 ng / ml). PRP significantly increased ALP inducing activity in a dose-dependent manner up to 1.25 μL / well (12.5 μL / well for activated PRP described below) in the presence of BMP4, but ALP inducing activity tended to decrease at higher doses. It was. As the amount of PRP added increases, the amount of growth factors such as platelet-derived PDGF and IGF increases, and cell proliferation becomes active and differentiation into osteoblasts is suppressed. Conceivable.

  As described above, human PRP alone did not exert any effect on the differentiation from mesenchymal cells to osteoblasts (mobilization of osteoblasts), but when used in combination with BMP, It was revealed that it has an excellent effect of remarkably enhancing the induction of differentiation into blast cells.

Next, it was examined whether bovine PRP has the same effect as human PRP. As in the case of human PRP, bovine PRP (platelet in the original plasma was concentrated about 100 times) was prepared from commercially available bovine plasma (Funakoshi). 0 to 5 μL of bovine PRP was added to each well (culture solution 150 μL / well) of a 96-well plate in which a myoblast cell line, C2C12, adipocyte precursor cell line, and MC3T3-L1 were cultured. However, it was found that PRP was coagulated and activated and promoted the growth of C2C12 cells. In addition, the addition of PPP also promotes the growth of C2C12 cells, but it was revealed that PRP has a higher proliferation promoting activity. When PRP is added to the cell culture medium, PRP is coagulated and activated, and even when PPP is added, a fibrin clot is formed by Ca ²⁺ ions in the medium. Compared to human-derived PRP and PPP, bovine-derived PRP and PPP have a stronger fibrin clot, and the fibrin clot was removed when the culture solution was removed or the cells were washed to measure the number of cells or ant-kariphosphatase activity. The cell layer tended to peel off with removal. Therefore, as described in Example 2, bovine PRP (prepared so that the platelet count is about 100 times that of the original plasma as in the case of human PRP described above) and the PPP of its supernatant were each calcium chloride. And activated bovine thrombin were used to prepare activated PRP (activated PRP) and activated PPP (activated PPP). Since PRP is diluted 10-fold by the activation treatment of PRP, the activated PRP corresponds to the platelet count of the original plasma concentrated about 10-fold. Similarly for activated PRP and activated PPP from which platelet activation and fibrin clot were generated in advance by calcium chloride and thrombin treatment, and then cleaved (3,000 rpm, 10 minutes) to remove fibrin clot. The proliferation activity and ALP induction activity of the cell line were measured. As shown in Example 2, activated PRP (addition amount was 10 times the original PRP) promoted the growth of C2C12 cells, which are mesenchymal cells, and the growth promoting activity was almost equivalent to the original PRP. It was. This indicates that the promotion of cell proliferation by PRP is due to platelet-derived growth factors that are abundant in PRP. In fact, it has already been clarified that the cell growth activity of PRP is due to growth factors such as platelet-derived PDGF, IGF and VEGF (Boyapati. L. and Wang HL .: Implant Dentistry 15: 160-168 , 2006).

  In order to promote bone formation and bone regeneration, it is necessary to enhance differentiation induction from mesenchymal cells to osteoblasts (recruitment of osteoblasts) and promote differentiation and maturation of osteoblast precursor cells and osteoblasts. is there. Therefore, the present inventors used the adipocyte progenitor cell line, MC3T3-L1, as a mesenchymal cell line that is more difficult to differentiate into the myoblast cell line, C2C12 and osteoblasts than the C2C12 cell as a mesenchymal cell. The activity of inducing differentiation into osteoblasts by activated PRP was examined. Differentiation from mesenchymal cells to osteoblasts by activated PRP was measured using the induction activity of alkaline phosphatase (ALP) as an index. As shown in Example 2, activated PRP alone does not show ALP-inducing activity against any mesenchymal cell lines (C2C12 and MC3T3-L1), and induces differentiation of mesenchymal cells into osteoblasts. It became clear that he did not have the ability. However, it was found that activated PRP strongly enhances differentiation induction from C2C12 cells to osteoblasts when used in combination with a low concentration of BMP4 (25 ng / ml). MC3T3-L1 has low sensitivity to BMP, and differentiation into osteoblasts required a relatively high concentration of BMP4 (800 ng / ml) (it is difficult to differentiate into osteoblasts). (Suggested), activated PRP remarkably enhanced differentiation induction from MC3T3-L1 cells to osteoblasts by BMP4.

  Promotion of osteogenesis requires the promotion of differentiation and maturation of osteoblast progenitor cells and osteoblasts, and research has been conducted energetically. Then, ST2 and MC3T3-E1 cells were used as osteoblast precursor cell lines, and ALP-inducing activity by activated PRP was examined. As shown in Example 3, activated PRP showed higher growth promoting activity than activated PPP with respect to the growth of ST2 cells. Regarding differentiation / maturation of these osteoblast progenitor cells into osteoblasts, the effect of inducing ALP production in ST2 cells and MC3T3-E1 cells was not observed with activated PRP alone. As shown in Example 3, the combined use of a constant amount of activated PRP (12.5 μL / well) per well (culture medium 150 μL) and various concentrations of BMP4 allows the ALP production inducing ability of ST2 cells and MC3T3-E1 cells to be increased. Enhanced.

  As described above, PRP is not only used for induction of differentiation from mesenchymal cells to osteoblasts (mobilization of osteoblasts) but also for osteoprogenitor progenitor cells and osteoblasts when applied alone as in the prior art. Since no effect on differentiation / maturation is manifested, it was not possible to scientifically prove the effect of PRP on promoting bone formation / regeneration as in the previous literatures disclosed so far. However, the present inventors have found that the combined use of PRP and BMP is (1) induction of differentiation from mesenchymal cells to osteoblasts (mobilization of osteoblasts) and (2) differentiation of osteoblast precursor cells and osteoblasts.・ It has been found that it exhibits an excellent effect that satisfies the conditions necessary for bone formation and bone regeneration promotion to remarkably enhance maturity.

  Cells committed to differentiation in the osteoblast line are highly sensitive to BMP (<10 ng / ml), and low concentrations of BMP are known to promote differentiation and maturation of osteoblasts. Cells committed to differentiation into mesenchymal cells have low sensitivity to BMP (> 100 ng / ml), and it is difficult to induce mesenchymal cells to differentiate into osteoblasts with BMP alone. This is considered to be one of the reasons why BMP cannot be used as a drug for osteogenesis. In addition, PRP does not have the activity to induce differentiation of mesenchymal cells into osteoblasts and the activity to promote differentiation and maturation of osteoblasts, so it is difficult to expect as an agent for promoting bone formation and bone regeneration. It is. However, the combined use of PRP and BMP strongly enhances (1) differentiation induction from mesenchymal cells to osteoblasts (mobilization of osteoblasts) and (2) osteoblast precursor cells to osteoblasts. Since it promotes differentiation and maturation into cells, it is expected as an agent for promoting bone formation and bone regeneration.

  BMP is a protein belonging to the TGF-β spur family, and to date, nearly 20 BMP members have been identified. Among them, BMP2, BMP4, BMP6, BMP7 (OP-1) and BMP9 are known to be deeply involved in bone metabolism and bone formation (Cheng H., et al .: J. Bone and Joint Surgery ( American) 85: 1544-1552, 2003). Examples 1 and 2 show a remarkable enhancement effect by the combination of human or bovine PRP and BMP4 in the induction of differentiation from mesenchymal cells to osteoblasts. It was investigated whether the same effect was expressed by combined use. As shown in Example 4, BMP2, BMP6, and BMP7 (OP-1) are also induced to differentiate from myoblast cell lines, which are mesenchymal cells, and C2C12 to osteoblasts, in combination with PRP or activated PRP. Strongly enhanced and universality was recognized.

  For fracture treatment and bone regeneration in bone loss or defect, treatment by transplantation of a stent device or an artificial bone prosthetic material is performed. For example, for the treatment of fractures in the orthopedic field, a collagen stent was used as a carrier (as a scaffold for cell adhesion and proliferation, as scaffold), and a stent device for transplantation that was impregnated with BMP2 or BMP7 (OP-1) was developed. ing. In the dental field, alveolar bone regenerative medicine is performed by the GBR (guided bone regeneration) method using PRP in combination with porous β-TCP mainly composed of calcium phosphate and hydroxyapatite as an artificial bone prosthesis material prior to implantation. It has been broken. These use BMP and PRP alone for each carrier, and the former has been found in clinical trials to significantly reduce the treatment period (hospitalization period) compared to autologous bone transplant, which is the gold standard. Moreover, despite the local administration, the dose is relatively high (several mg / device). As for the latter PRP, as described above, many contradictory results have been reported on the bone formation and bone regeneration promoting effects of PRP, and there is no scientific evidence.

As shown in Examples, the combined use of PRP and BMP of the present invention is (1) induction of osteoblast differentiation from mesenchymal cells (mobilization of osteoblasts) and (2) differentiation and maturation of osteoblasts. Show remarkable effects. Therefore, whether BMP and PRP-derived active ingredients are both adsorbed and retained on a porous artificial bone filling material mainly composed of collagen sponge or calcium phosphate, and a remarkable combined effect of BMP and PRP is exhibited. I checked. In BMP4 and activated PRP using collagen sponge, a scaffold required for cell adhesion and proliferation, and porous hydroxyapatite (CELLYARD HA scaffold) with the same quality and properties used in medicine We examined whether or not the active ingredients in the sorbent could be adsorbed and retained by these scaffolds. As shown in Example 5, a constant amount of BMP4 (3 ng / collagen sponge or 5 ng / hydroxyapatite) and activated PRP in various amounts (0, 12.5, 25 and 50 μL / collagen sponge or hydroxy as shown in Example 5) Apatite) and allowed to stand at room temperature for about 1 hour for adsorption. Subsequently, each carrier was washed three times with shaking with sterile PBS, and then C2C12 cells were inoculated into each carrier and cultured at 37 ° C. for 6 to 7 days. After discarding the culture solution and washing 3-4 times with PBS, the ALP activity of C2C12 cells grown on each carrier was measured. As shown in Example 5, the ability of C2C12 cells grown on a collagen sponge carrier to induce ALP production was significantly enhanced by the combined use of BMP4 and active PRP rather than BMP4 alone. This means that both BMP4 and activated PRP-derived active ingredients are adsorbed and stably retained on the collagen sponge without being washed away even with sufficient washing with PBS, thereby enhancing differentiation induction from mesenchymal cells to osteoblasts. It is shown that. Of particular interest is the amount of PRP added to the minimum BMP4 concentration (20-25 ng / ml) that can induce ALP activity with BMP alone when added to a 150 μL / well culture medium (in the case of the culture medium). On the other hand, the amount of activated PRP (corresponding to the concentration of platelets about 10 times that of the original plasma) was 1 to 33%, particularly preferably 2%, per well (150 μL) of culture medium per well. Whereas ~ 17% (Examples 1-5), collagen sponge (200 mm ² x 1 mm, volume 200 mm ³ ) with 60 ng / ml BMP4 50 μL (3 ng / sponge) and activated After PRP (0-50μL / sponge) is adsorbed and retained, when cultivated in 2 mL of culture solution in each well of a 24-well plate, the amount of activated PRP added relative to the amount of culture solution (2 mL) However, the effectiveness of the activated PRP was confirmed at an addition amount of about 3 to 25% with respect to the collagen sponge volume (200 mm ³ ) (Example 5). Within the range of addition amount It was there. This suggests that a stent device for transplantation using a collagen sponge as a carrier can be applied in the range of 1 to 33.3%, particularly preferably 2 to 17%, based on the transplantation volume. However, the range of the above addition amount is applicable when the active ingredient derived from BMP4 and PRP shows a very high adsorption / retention rate (90% or more) like the collagen sponge used in Example 5. As in the case of porous hydroxyapatite described later, since the adsorption / retention rate varies depending on the material and structure (total surface area per volume), the above range of addition amount must be corrected based on the adsorption / retention rate. . As shown in Example 5, even when porous hydroxyapatite (HA) was used as the carrier, both the BMP4 and PRP-derived active ingredients were adsorbed and retained on the carrier as in the case of the collagen sponge. In combination, the differentiation induction from C2C12 cells to osteoblasts was remarkably enhanced. However, there is a tendency that the adsorption / retention rate of the active ingredient derived from PRP is worse than that of the collagen sponge, and in order to adsorb and retain an effective amount of the PRP component, it is about twice or more than that of the collagen sponge per HA volume. Addition amount was required. The reason why the adsorption / retention rate by HA is inferior to that of collagen sponge is considered to be due to the characteristics of HA or due to the slow adsorption rate.

  Based on the above results, a group consisting of artificial bone prosthetic materials mainly composed of calcium phosphate, such as collagen sponge, β-TCP, porous hydroxyapatite (HA), etc., which are currently used in transplanted stent devices for fracture treatment. It can be transplanted stably to the affected area by impregnating (supporting) a combination of PRP (or activated PRP) and BMP.

Effects of the present invention

  According to the present invention, when PRP and BMP are used in combination, (1) powerful enhancement of differentiation from mesenchymal cells to osteoblasts (recruitment of osteoblasts) and (2) bones of osteoblast precursor cells Differentiation into blast cells and differentiation / maturation of the osteoblasts are promoted. In addition, a combination agent of PRP and BMP is carried in one selected from the group consisting of an artificial bone filling material (for example, β-TCP, porous hydroxyapatite, etc.) mainly composed of collagen sponge or calcium phosphate, and is applied to the affected area. By transplanting, it is possible to effectively prevent and treat bone diseases requiring bone formation / bone regeneration due to fracture treatment or bone loss, particularly alveolar bone regeneration.

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. Various changes and modifications can be made to the present invention, and these are also included in the scope of the present invention.

Example 1: Effect of enhancing differentiation induction from mesenchymal cell line C2C12 by combined use of PRP and BMP4 prepared from fresh blood of healthy humans Using sodium citrate as an anticoagulant, healthy humans The fresh blood collected from the sample is aseptically subjected to first-stage centrifugation (900 xg, 10 minutes) to prepare platelet-containing plasma. About half of the fresh blood plasma is subjected to the second-stage centrifugation (2,100 xg, 20 minutes), and the platelet fraction concentrated and precipitated at the bottom of the centrifuge tube is a small amount of supernatant plasma (PPP The platelet rich plasma (PRP) was prepared so as to be 1/100 volume of the original plasma volume (PRP platelets were concentrated to about 100 times that of the original plasma). The second-stage centrifugation is preferably performed under such conditions that platelets in plasma can be collected in PPR as much as possible.

The myoblast cell line C2C12, which is a mesenchymal cell line, was cultured in a DMEM containing 15% fetal bovine serum (FBS) until approximately 90% confluent using a 75 T-flask for adherent cells. The cells were detached with a 0.25% trypsin solution containing 1 mM EDTA, and suspended in DMEM containing 1% FBS so that the number of cells became 2 × 10 ⁵ / ml. 50 μL was added to each well of a 96-well plate (IWAKI) coated with type I collagen, and cultured overnight in a 37 ° C. CO ₂ incubator.

The culture medium in each well was removed, and then 50 μL each of serum-free DMEM containing penicillin A (100 u / ml) and streptomycin (0.1 mg / ml) was added to each well. Add 0, 0.625, 1.25, 2.5 and 5 μL of the human PRP prepared above to each well in triplicate (n = 3) to each well, and then adjust the plasma concentration in all wells to 10 μL / well. The human PPP was added to each well. Furthermore, serum-free DMEM was supplemented with 40 μL of each well, and the amount of medium in each well was adjusted to 100 μL. Finally, 50 μL of serum-free DMEM containing 60 ng / ml BMP4 was added to each well (total medium volume: 150 μL / well; final concentration of BMP4: 20 ng / ml). In the BMP4 non-added group, 50 μL of serum-free DMEM was added to each well. It was confirmed that PRP was coagulated and activated by adding it to the cell culture medium. The 96-well plate thus prepared was cultured for 6 days in a 37 ° C. CO ₂ incubator. After culturing, the culture solution is removed, and the cells are washed twice with PBS (Takara Bio), and then 100 μL of ethanol: acetone (1: 1) mixture is added to each well, and the cells in each well are fixed at room temperature for about 1 minute. Turned into. Remove the ethanol: acetone mixture, air dry, and add 100 μL of a buffer solution (0.1 M diethanolamine, 1 mM MgCl ₂ , pH 10) containing 1 mg / ml p-nitrophenyl phosphate to each well. And allowed to react at room temperature for 20 minutes. After stopping the reaction by adding 50 μL of 3M NaOH to each well, alkaline phosphatase activity was measured by measuring the absorbance at 405 nm of each well.

  The enhancement effect of human PRP on the differentiation induction from C2C12 cells to osteoblasts by BMP4 is shown in FIG. Human PRP has no ability to induce ALP production in the absence of BMP, but in the presence of a small amount of BMP4 (20 ng / ml), 0.625 (0.42%) to 5 μL for each well culture medium (150 μL) It was found that the differentiation from C2C12 cells to osteoblasts was strongly enhanced with the addition range of (3.3%). In particular, the enhancement effect was the highest when 0.625 and 1.25 μL were added, and the enhancement effect tended to decrease when the addition was further increased. This is thought to be due to the fact that proliferation increased due to an increase in growth factors such as platelet-derived PDGF and IGF as the amount of PRP added increased, and conversely, differentiation was suppressed.

Example 2: Effect of enhancing differentiation induction from mesenchymal cell lines C2C12 and MC3T3-L1 to osteoblasts by the combined use of PRP and BMP4 prepared from bovine plasma Figure 1 shows the use of PRP other than human-derived PRP in combination with BMP4. We investigated whether the induction of differentiation from mesenchymal cell line C2C12 into osteoblasts was enhanced. Platelets are concentrated and precipitated from commercially available bovine plasma (Funakoshi) by centrifugation corresponding to the second step described in Example 1, and reconstituted to bovine PPP that is 1/100 of the original plasma volume as in the case of human PRP. Bovine PRP was prepared by suspending.

Since addition of human PRP was observed to promote the growth of C2C12 cells as described in Example 1, the number of cells in each well was determined using the Cell Count Reagent SF kit (Nacalai tesque) in addition to alkaline phosphatase activity. And measured. Bovine PRP or bovine PPP was added instead of human PRP in Example 1, and after 4 days of culture, the culture medium in each well was removed, and the cells were washed with PBS, and then 100 μL of PBS was added to each well. 10 μL of Cell Count Reagent SF kit was added to each well and left in a CO ₂ incubator at 37 ° C. for 1-2 hours. Soluble formazan produced by reduction by cells was measured by absorbance at 492 nm.

Bovine PRP and PPP-derived fibrin clots formed in each well when bovine PRP or PPP is added after 4 days of culturing are removed are stronger than those derived from human PRP. There was a tendency for the cells to peel off with the removal of the fibrin clot. Therefore, before adding bovine PRP or PPP to the culture broth, a fibrin clot was formed in advance by platelet activation treatment, and then activated PRP and activated PPP were prepared by removing the fibrin clot by centrifugation. . To activate PRP, first mix 88 μL of medium (DMEM or αMEM) containing 1% FBS as an activator, 32 μL of bovine thrombin (WAKO) solution (0.5 u / μL), 40 μL of 1% CaCl ₂ (total 160 μL). Prepared. Next, 160 µL of the above activator was added to a mixture of PRP or PPP 160 µL and 1% FBS-containing medium (DMEM or αMEM) 1280 µL (total 1600 µL), and immediately stirred to activate platelets in PRP and fibrin coagulation A lump is formed. Collect and remove the fibrin clot by centrifugation (3,400 rpm, 10 minutes). The activated PRP prepared in this way is a 10-fold dilution of PRP and corresponds to PRP in which the platelets in the original plasma are concentrated about 10-fold. Similarly, activated PPP from which the fibrin clot was removed was prepared.

As described in Example 1, 96 C2C12 cell suspension (50 μL, 2 × 10 ⁵ / ml) suspended in DMEM containing 1% FBS was added to each well and cultured overnight. -Add 0, 6.25, 12.5, 25 and 50 μL of activated PRP or activated PPP to each well of triplicate (n = 3) well plate (IWAKI), and then add 100 μL of medium in each well. Was supplemented with DMEM containing 1% FBS. Next, 50 μL of BMP4 solution (75 ng / ml) diluted with DMEM containing 1% FBS was added to each well (final medium volume was 150 μL / well, final concentration of BMP4 was 25 ng / ml). In the BMP4 non-added group, 50 μL of DMEM containing 1% FBS was added to each well. The 96-well plate thus prepared was further cultured for 4 days in a CO ₂ incubator. First, in order to measure the number of cells in each well, the plate to which the Cell Count Reagent SF kit was added as described above was held at 37 ° C. for 1 to 2 hours, and the absorbance at 492 nm of each well was measured. The result is shown in FIG. Further, Cell Count Reagent in each well of the same plate where the number of cells was measured was washed and removed twice with PBS, and then the alkaline phosphatase activity in each well was measured according to the method described in Example 1. The alkaline phosphatase activity per cell number (OD. 492 nm = 1.00) in each well is shown in FIG. Furthermore, FIG. 4 shows the enhancing effect of activated PRP in the differentiation induction from C2C2 cells to osteoblasts by the low concentration region of BMP4. As shown in FIG. 2, the addition of bovine-derived activated PRP or activated PPP promotes the growth of C2C12 cells, but it can be seen that activated PRP has a stronger proliferation promoting activity. This suggests that platelet-derived growth factors such as PDGF and IGF that are abundant in PRP are involved in promoting the growth of C2C12 cells. The tendency that the cell growth promoting activity by activated PRP slightly decreases with the addition of BMP4 is considered to be due to the enhanced differentiation into osteoblasts due to the coexistence of PRP and BMP4.

  Alkaline phosphatase activity per number of cells in each well was determined. As shown in Fig. 3, almost no alkaline phosphatase activity was observed even when activated PRP was added in the group without BMP4, and PRP alone was mesenchymal. It shows that it has no activity to induce differentiation of cells into osteoblasts. Differentiation induction from C2C12 cells to osteoblasts is strongly enhanced by the combination of bovine activated PRP and BMP4 over the combination of bovine activated PPP and BMP4, and bovine PRP has the same activity as human PRP Was recognized. Activated PRP is added from C25C12 cells to osteoblasts by adding 6.25 (4.2%) to 50 μL (33%) per well (150 μL culture medium) in the presence of a small amount of BMP4 (25 ng / ml). Strongly enhanced the differentiation. In particular, the enhancement effect tended to increase dose-dependently up to 25 μL per well and decreased with the addition of 50 μL. As in the case of human PRP, this suggests that platelet-derived growth factors are increased by increasing the dose of activated PRP, C2C12 cells are actively proliferating, and differentiation is relatively suppressed. ing. On the other hand, when BMP4 (25 ng / ml) and activated PPP were used in combination, alkaline phosphatase activity tended to increase gradually with weak cell growth promotion due to the increase in the amount of activated PPP added. Regardless of the amount of added PPP, almost constant alkaline phosphatase activity was exhibited (FIG. 3). That is, activated PPP shows that BMP4 does not enhance differentiation induction from C2C12 cells to osteoblasts. In addition, as shown in FIG. 4, activated PRP strongly enhanced the weak differentiation induction from C2C12 cells to osteoblasts by a low concentration of BMP4.

  Myoblast cell line C2C12 has been reported to be induced to differentiate into osteoblasts by high concentration (300 ng / ml) of BMP2 (Katagiri T. et. Al .: J. Cell Biology 127: 1755-1766, 2004), and is often used in research on differentiation induction from mesenchymal cells to osteoblasts. We investigated whether the enhancement of differentiation induction from mesenchymal cells to osteoblasts by the combined use of PRP and BMP4 is also exerted on mesenchymal cells other than C2C12. Among mesenchymal cells, the adipocyte progenitor cell line MC3T3-L1, which is difficult to induce differentiation into osteoblasts compared to C2C12 cells, was used.

MC3T3-L1 cells were cultured in αMEM medium containing 10% FBS until they reached about 90% confluence, then trypsinized as described in Example 1, and the number of cells was 1 × using αMEM containing 10% FBS. A 10 ⁴ / ml MC3T3-L1 cell suspension was prepared. 50 μL of this cell suspension was added to each well of a 96-well plate (IWAKI) coated with type I collagen and cultured in a CO ₂ incubator for 2 days. After culturing, the culture solution was removed, and 50 μL of αMEM containing 1% FBS was added to each well. Add 0, 6.25, 12.5, 25 and 50 μL of bovine-derived activated PRP or PPP to each well of triplicate (n = 3), and then add 1% FBS so that the volume of medium in each well is 100 μL. Supplemented with αMEM. Finally, 50 μL of αMEM containing 2400 ng / ml BMP4 and 1% FBS was added to each well (final medium volume 150 μL / well; final BMP4 concentration 800 ng / ml). In the BMP4 non-added group, 50 μL of αMEM containing 1% FBS was added to each well. The 96-well plate thus prepared was further cultured for 6 days in a CO ₂ incubator. The number of cells and alkaline phosphatase activity in each well were measured as described in Example 2. The alkaline phosphatase activity (OD.405 nm / OD.492 nm) per cell number (OD.492 nm) in each well is shown in FIG. For MC3T3-L1, neither activated PRP nor activated PPP showed a cell growth promoting effect. However, activated PRP was found to significantly enhance the induction of MC3T3-L1 cell differentiation into osteoblasts by BMP4 compared to activated PPP (FIG. 5). The enhancement effect by activated PRP was observed in the range of 6.25 μL (4.2%) to 50 μL (33.3%) added per well (150 μL of culture medium).

  As described above, not only C2C12 cells but also adipocyte precursor cell line MC3T3-L1, which is relatively difficult to induce differentiation into osteoblasts among mesenchymal cells, PRP induces differentiation into osteoblasts by BMP4. Remarkably enhanced.

Example 3: Differentiation / maturation promoting effect of osteoblast precursor cell lines ST2 and MC3T3-E1 into osteoblasts by combined use of PRP and BMP4 Using ST2 and MCT3-E1 cells as osteoblast precursor cell lines, bovine The effect of promoting the differentiation and maturation of osteoblasts by the combined use of activated PRP derived from BMP4 was investigated.

ST2 and MC3T3-E1 cells are cultured in αMEM containing 10% FBS until reaching about 90%, trypsinized as described in Example 1, and cells of ST2 and MC3T3-E1 are used using αMEM containing 10% FBS. Each cell suspension was prepared so that the number was 1 × 10 ⁴ / ml. 50 μL of each cell suspension was added to each well of a 96-well plate coated with type I collagen and cultured in a CO ₂ incubator for 2 days. After the culture, the culture solution was removed, and 50 μL of αMEM containing 1% FBS was added to each well. Next, the amount of activated PRP added was 12.5 μL (8.3%) per well (culture medium 150 μL) in which a high potentiating effect was always observed within the range of the effective amount of activated PRP revealed so far. ) Was added to each well, and 12.5 μL of activated PPP was added to each well for the control group. Each well was supplemented with 35 μL of αMEM containing 1% FBS, and the medium volume was adjusted to 100 μL / well. Subsequently, 50 μL of BMP4 solution (18.6 to 300 ng / ml) serially diluted with αMEM containing 1% FBS was added to each well of a 96-well plate of ST2 cells (final concentration of BMP4 was 6.2 to 100 ng / ml). ). On the other hand, 50 μL of BMP4 solution (9.4 to 600 ng / ml) serially diluted in the same manner was added to each well of a 96-well plate of MC3T3-E1 cells (final concentration of BMP4 was 3.13 to 200 ng / ml). In the BMP4 non-added group, 50 μL of αMEM containing 1% FBS was added to each well. The 96-well plate thus prepared was cultured in a CO ₂ incubator at 37 ° C. for 6 days. As described in Example 2, the number of cells and alkaline phosphatase activity in each well were measured, and the alkaline phosphatase activity (OD. 405 nm / OD. 492 nm) per cell number (OD. 492 nm = 1.0) was determined. It was. FIG. 6 shows the effect of promoting the differentiation / maturation of ST2 into osteoblasts by the combined use of activated PRP and BMP4. Activated PRP strongly enhanced differentiation and maturation of ST2 cells into osteoblasts when the amount of BMP4 added was 25 ng / ml or more, compared to activated PPP.

  FIG. 7 shows the effect of promoting the differentiation / maturation of MC3T3-E1 cells into osteoblasts by the combined use of activated PRP and BMP4. The combination of activated PRP and BMP4 tended to enhance the ability of MC3T3-E1 cells to induce ALP production at all doses of BMP4 compared to the combination of activated PPP and BMP4. However, the ability to induce ALP production was significantly enhanced at 50 ng / ml or more. This indicates that the combined use of activated PRP and BMP4 promotes the differentiation and maturation of MC3T3-E1 cells into osteoblasts.

  As described above, it was confirmed that the combined use of activated PRP and BMP promoted differentiation / maturation of osteoblast precursor cells into osteoblasts. However, the combined effect of activated PRP and BMP4 on osteoblast progenitor cell lines is not as pronounced as that of mesenchymal cell lines C2C12 and MC3T3-L1. This is thought to be because the cells are committed to differentiation, and are sensitive to BMP and easily differentiate into osteoblasts even with BMP alone.

Example 4: Effect of enhancing differentiation induction from mesenchymal cells to osteoblasts by the combined use of PRP and BMP members other than BMP4 Among nearly 20 types of BMP members, BMP2, BMP4, BMP6, BMP7 (OP- 1) and BMP9 have been shown to be deeply involved in bone metabolism and bone formation (Cheng H., et al .: J. Bone and Joint Surgery (American) 85: 1544-1552, 2003). Therefore, BMP2 (Acris Antibodies GmbH, Germany; Host: E. coli; Specific activity: Mouse chondrocyte progenitor cell line, ATDC-5 cell ALP production inducing ability ED50 = 0.5-1.0 μg / ml), BMP6 (R & D Systems, host: mouse myeloma cell line NSO cells; specific activity: ADC production inducing ability of ATDC-5 cells with ED50 = 0.05-0.15 μg / ml) and BMP7 (OP-1) (R & D System Host: CHO cells; Specific activity: ADC production-inducing ability of ATDC-5 cells (ED50 = 0.2-0.6 μg / ml), and using activated PRP derived from bovine in the same manner as described in Example 2. The effect of enhancing differentiation induction from C2C12 cells to osteoblasts was investigated.

The effect of promoting the proliferation of C2C12 cells by the combined use of activated PRP derived from bovine and BMP2 is shown in FIG. 8, and the effect of enhancing differentiation induction from C2C12 cells to osteoblasts is shown in FIG.
FIG. 10 shows the growth promotion effect of C2C12 cells by the combined use of activated PRP and BMP6, and FIG. 11 shows the enhancement effect of differentiation induction from C2C12 cells to osteoblasts.

FIG. 12 shows the effect of promoting the proliferation of C2C12 cells by the combined use of activated PRP and BMP7 (OP-1), and FIG. 13 shows the effect of enhancing differentiation induction from C2C12 cells to osteoblasts.
The production host of BMP2 was Escherichia coli, the specific activity was the lowest among the BMP members used this time, and alkaline phosphatase was gradually induced in C2C12 cells at 300 ng / ml. Thus, the effect of promoting cell proliferation and enhancing osteoblast differentiation induction by using BMP2 (300 ng / ml) in combination with activated PRP was examined. Although the proliferation of C2C12 cells was most promoted by adding activated PRP alone, the proliferation promoting effect was reduced by the combined use with BMP4 (FIG. 8). On the other hand, in the combined use of activated PRP and BMP2, activation induction was strongly enhanced in the range of 6.25 (4.2%) to 50 μL (33.3%) per well (culture medium 150 μL). In particular, the enhancement effect was most remarkable in the range of 6.25 (4.2%) to 25 μL (16.7%) (FIG. 9).

  The growth promoting activity of C2C12 cells by the combined use of activated PRP and BMP6 was almost the same as that of activated PRP alone (FIG. 10)). The amount of activated PRP added in the range of 6.25 (4.2%) to 25 μL (16.7%) per well (150 μL culture medium) strongly enhanced differentiation induction from C2C12 cells to osteoblasts by BMP6 (FIG. 11).

  The proliferation-promoting activity of C2C12 cells by the combined use of activated PRP and BMP7 (OP-1) was lower than that obtained by adding activated PRP alone (FIG. 12). On the other hand, the addition amount of activated PRP strongly enhanced differentiation induction from C2C12 cells to osteoblasts by BMP7 in the range of 6.25 (4.2%) to 25 μL (16.7%) per well (culture medium 150 μL) (FIG. 13). ).

  As described above, not only BMP4 but also BMP2, BMP6 and BMP7 (OP-1) can be used together with activated PRP in the same amount range (4.2 to 16.7%) to convert C2C12 cells to osteoblasts. It became clear that differentiation induction was remarkably enhanced.

Example 5: Examination of a carrier that adsorbs and retains both BMP and activated PRP-derived active ingredients and efficiently exhibits the combined effect of both
We investigated a carrier (scaffold) that adsorbs and retains both BMP and activated PRP-derived active ingredients, and efficiently and significantly enhances differentiation induction from mesenchymal cells to osteoblasts.

First, as a carrier, sterile collagen sponge (surface area is about 200 mm ² , thickness is 1 mm, volume is about 200 mm ³ ) is placed in each well of 24-well plate for floating cells, and BMP4 solution (60 ng) prepared in serum-free DMEM / ml) was soaked in 50 μL (3 ng BMP4), then activated PRP was soaked in 12.5, 25 and 50 μL, respectively, and allowed to adsorb to the collagen sponge by shaking at room temperature for 1 hour. The control group includes (1) no BMP4 and activated PRP added, (2) no BMP4 added, 12.5 μL soaked activated PRP, and (3) no BMP4 solution added with activated PRP. What was impregnated with 50 μL (3 ng BMP4) was prepared. After adsorption at room temperature for 1 hour, all collagen sponges were thoroughly washed with shaking 3 times with sterile PBS to completely remove PBS. Next, add 1 ml of C2C12 cell suspension prepared with DMEM containing 1% FBS to 2 x 10 ⁵ / ml to each well containing collagen sponge and incubate for 2 hours in a CO ₂ incubator. After the cells were adhered to the collagen sponge, 1 ml of DMEM containing 1% FBS was further added to each well (2 ml / well in total). The 24-well plate containing the collagen sponge thus prepared was further cultured for 7 days in a CO ₂ incubator. After culturing, the culture solution in each well was removed, and the collagen sponge in the well was washed 4 times with 2 ml of PBS. 500 μL of alkaline phosphatase substrate solution described in Example 1 was added to each well and allowed to react at room temperature for 20 minutes. 100 μL was collected from each well of a 24-well plate, transferred to a 96-well plate, 50 μL of 3M NaOH was added to stop the reaction, and the absorbance at 405 nm of each well was measured. The results are shown in FIG. Almost no alkaline phosphatase activity was detected in the collagen sponge adsorbed without addition of BMP4 and activated PRP in the control group, and added with 12.5 μL of activated PRP without addition of BMP4. As shown in FIG. 14, in the group to which BMP4 and activated PRP were added in combination, activated PRP was added in the range of 12.5 (6.3% per collagen sponge volume) to 50 μL (25%) compared to BMP4 alone. A significant enhancement effect of differentiation induction from cells to osteoblasts was observed. Among them, the enhancement effect was highest in the group with 12.5 μL (6.3%) of activated PRP. As described above, both BMP4 and active PRP-derived active ingredients are strongly adsorbed and retained on the collagen sponge so that they cannot be washed away even with sufficient PBS washing, and the effect of significantly enhancing osteoblast differentiation induction from mesenchymal cells It became clear to express. The effective addition amount of activated PRP is in the range of 6.3-25% per collagen sponge volume, and is in the range of 4.2-33.3%, which is the effective addition amount of activated PRP per culture solution in Example 2. It was. Like the collagen sponge used in this example, in the case of an artificial bone filling material or a stent device for transplantation that can efficiently adsorb and retain both PRP-derived active ingredients and BMP4 (90% or more), It indicates that the amount of active PRP added is preferably in the range of 4.2 to 33.3% with respect to its volume.

Porous hydroxyapatite (CELLYARD HA) has the same porosity and properties as an artificial bone prosthetic material actually used in medicine. Using this porous hydroxyapatite (HA, diameter 5 mm x 2 mm, 39 mm ³ ) as a carrier, each HA was placed in each well of a 96-well plate for floating cells, and a BMP4 solution (500 ng) prepared in serum-free DMEM. / ml) soaked in 10 μL (5 ng BMP4 / HA), allowed to stand at room temperature for 1 hour, then added activated PRP per HA to 12.5 25 and 50 μL, respectively, and shaken at room temperature for 1 hour to adsorb to HA It was. The control group was soaked with 10 μL (5 ng BMP4) of BMP4 solution (500 ng / ml) without (1) BMP4 added, activated PRP added, and (2) no activated PRP added. Things were prepared. After adsorption at room temperature for 1 hour, all HA was thoroughly washed with 150 μL of sterile PBS for 10 minutes for a total of 3 times, and then PBS was completely removed. Next, add 50 μL of C2C12 cell suspension prepared in MEM containing 10% FBS to 2 x 10 ⁵ / ml to each well containing HA, and incubate for 2 hours in a CO ₂ incubator. After adhering the cells to HA, 100 μL of DMEM containing 1% FBS was further added to each well (total 150 μL / well). The plate thus prepared was cultured at 37 ° C. for 6 days. The alkaline phosphatase activity in each well is shown in FIG.

  As shown in FIG. 15, even when BMP4 alone was added, differentiation induction from C2C12 cells to osteoblasts was promoted by HA culture. In the combination addition group of BMP4 and active PRP, differentiation enhancement from C2C12 cells to osteoblasts showed the highest enhancement effect when the amount of activated PRP added was 12.5 μL (31.8% per HA volume). Further, the enhancement effect was suppressed at 25 μL (63.6%), and no effect was observed at 50 μL (127.2%). The addition of active PRP to the culture broth tends to suppress the potentiating activity at 33.3% or higher, so the addition of 25 μL to HA (63.6%) is at least the maximum addition in the culture broth (33.3% ) It is estimated that it corresponds to the above. That is, under the adsorption / retention conditions used in this example, the adsorption of BMP4 to HA is efficiently adsorbed / retained to HA as in the case of collagen sponge, but the active ingredient derived from activated PRP to HA Adsorption / retention is inferior to collagen sponge, suggesting that more than twice the amount of activated PRP needs to be added. Thus, since the adsorption / retention ability of the active ingredient derived from BMP4 or PRP varies depending on the type, nature, structure, etc. of the carrier, the addition amount of both needs to be corrected by the adsorption / retention rate.

The effect of enhancing differentiation induction from mesenchymal cell line C2C12 to osteoblasts by the combined use of human platelet-rich plasma (PRP) and BMP4 is shown. The effect on the growth of bovine PRP, PPP and BMP4 mesenchymal cell line C2C12 is shown. The effect of enhancing differentiation induction from a mesenchymal cell line C2C12 to osteoblasts by the combined use of PRP and BMP4 is shown. It shows the differentiation-inducing enhancement activity from mesenchymal cell line C2C12 to osteoblast in the low BMP4 concentration region by PRP. The effect of enhancing differentiation induction from adipocyte progenitor cell line MC3T3-L1 to osteoblasts by the combined use of PRP and BMP4 is shown. The effect of promoting the differentiation / maturation of osteoblast progenitor cell line ST2 into osteoblasts by the combined use of PRP and BMP4 is shown. The effect of promoting the differentiation and maturation of osteoblast precursor cell line MC3T3-E1 into osteoblasts by the combined use of PRP and BMP4 is shown. The effect on proliferation of mesenchymal cell line C2C12 of PRP, PPP and BMP2 is shown. The effect of enhancing differentiation induction from mesenchymal cell line C2C12 to osteoblasts by the combined use of PRP and BMP2 is shown. The influence which it has on the proliferation of mesenchymal cell line C2C12 of PRP, PPP and BMP6 is shown. The effect of inducing differentiation of mesenchymal cell line C2C12 into osteoblasts by the combined use of PRP and BMP6 is shown. The effect on proliferation of mesenchymal cell line C2C12 of PRP, PPP and BMP7 is shown. The effect of inducing differentiation from a mesenchymal cell line C2C12 to osteoblasts in combination with PRP and BMP7 is shown. Fig. 4 shows the adsorption and retention of PRP-derived active ingredient and BMP4 by collagen sponge and the effect of enhancing differentiation induction from mesenchymal cell line C2C12 to osteoblasts. Adsorption / retention of PRP-derived active ingredient and BMP4 by porous hydroxyapatite (CELLYARD HA), and differentiation induction enhancing effect from mesenchymal cell line C2C12 to osteoblasts are shown.

Claims (7)

A method of promoting differentiation from mesenchymal cells to osteoblasts by using platelet rich plasma (PRP) and bone morphogenetic protein (BMP) in combination. A method of promoting differentiation / maturation of osteoblast precursor cells and / or osteoblasts by using platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP) in combination. In combination with platelet rich plasma (PRP) and bone morphogenetic protein (BMP), differentiation from mesenchymal cells to osteoblasts is promoted, and then osteoblast differentiation・ How to promote maturity. A preventive and therapeutic agent for bone diseases associated with fractures and bone loss, wherein the therapeutic agent comprises a combination of platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP). The platelet-rich plasma (PRP) -derived active ingredient and bone formation-inducing protein (BMP) are administered together with a carrier selected from the group consisting of a collagen sponge and an artificial bone filling material mainly composed of calcium phosphate. A prophylactic and therapeutic agent for bone diseases associated with fractures and bone loss. An osteoblast for prevention and treatment of bone disease accompanied by fracture and bone loss, obtained by culturing mesenchymal cells in the presence of platelet-rich plasma (PRP) and osteogenesis-inducing protein (BMP). A preventive and therapeutic agent for bone diseases accompanied by fracture and bone loss, comprising the osteoblast according to claim 6.