CN101249278B - Osteoinductive material and its preparation method and application - Google Patents

Abstract

The invention discloses a bone induction material which comprises bone induction factors and carriers; the bone induction factors comprise bone morphogenetic proteinbmp or basic fiberblast growth factors, and the carriers form gel matrix through the interaction among fibernogen, fibernectin, heparin, fiber protein stabling factor, thrombin and calcium chloride; the bone induction factor is embedded in the gel matrix. The invention further discloses a preparation method and application of bone induction material, which can release the bone morphogenetic proteinbmp slightly, efficiently and stably, and can improve the biological activity of bone morphogenetic proteinbmp.

Description

Osteoinductive material and its preparation method and application

【Technical field】

The invention relates to bone tissue repair materials, in particular to an osteoinductive material and its preparation method and application.

【Background technique】

Bone is next to blood the most common tissue to be repaired by transplantation. In the United States, more than 500,000 bone grafts are performed each year. Worldwide, up to 2.2 million bone grafts are performed annually in orthopedics, neurosurgery and dentistry to repair bone defects. The application in spine surgery accounts for about half of the total number of bone graft operations. The use of bone grafting in spine surgery is increasing rapidly as the population ages and the number of cases of degenerative spinal disease requiring treatment soars. Bone grafting is a surgical method used to treat bone defects that exceed the body's healing capacity and to perform bony fusion between vertebrae. In spinal surgery, the problem of maintaining spinal stability after spinal surgery is faced; implanting bone fusion materials between the vertebral bodies of the surgical segment or between the posterolateral transverse processes of the spine is an effective method to restore and maintain the long-term stability of the spine. The combined application of interbody fusion cage and bone graft material is the recommended operation for the treatment of single-segment spinal degenerative diseases. An intervertebral fusion cage is a hollow structure that is placed between adjacent vertebral bodies after the intervertebral disc is removed to stabilize the spine and restore the height between the vertebral bodies. The bone graft material is placed in the space in the middle of the intervertebral fusion cage, and the adjacent vertebral bodies are connected through bony healing to maintain the long-term stability of the spine.

Currently used bone graft materials include autologous bone, allogeneic bone and synthetic bone substitutes. Autograft bone is the gold standard of bone graft, but its source is limited, and it will cause new damage to the body when it is obtained; allograft bone has a wide range of sources, but it has the risk of immune rejection and disease transmission. These deficiencies above limit their wide application in clinic. In order to meet clinical needs, new synthetic bone substitute materials are constantly being developed and utilized. This includes natural materials, polymers, bioactive ceramics and several bone growth factors. Osteogenic growth factors are being extensively studied and applied for their powerful ability to induce or promote bone growth. Among them, bone morphogenetic proteins (bone morphogenetic proteins, BMPs) is the only growth factor known to induce osteogenic differentiation of stem cells and the most powerful growth factor for promoting osteogenesis. BMPs aqueous solution injected into the bone formation site in the body will be quickly cleared by the body, and effective bone formation cannot be achieved. Only when BMPs form a slow-release system with the carrier and maintain local continuous and effective release can the osteogenic effect be achieved. A slow-release system consisting of recombinant human bone morphogenetic protein-2 (rhBMP-2) and bovine absorbable collagen sponge (ACS) has been approved by the U.S. Food and Drug Administration (FDA) as an intervertebral fusion cage. Induced filler material for anterior lumbar surgery. The BMP-2/ACS slow-release system has been developed into a series of products by American Shufamo Company, which is put on the market under the trade name of INFUSE, and used together with the intervertebral fusion device (LT-cage) for anterior lumbar fusion. A large number of long-term clinical studies have shown that rhBMP-2/ACS slow-release system is significantly better than autologous bone graft for anterior lumbar fusion. This indicates that the combination of BMPs with appropriate carriers is expected to replace autologous bone as the new gold standard for bone grafting. However, the rhBMP-2/ACS sustained-release system also has obvious deficiencies. This slow-release system is released through passive simple diffusion in the body, and there is an initial burst release; this leads to insignificant controlled slow-release effects, a large amount of active factors used and wasted, and the scope of osteogenesis should not be limited. Due to the anatomical location, the use of the rhBMP-2/ACS sustained-release system in the lumbar spine has no obvious complications; however, the incidence of complications in other parts is quite high, manifested as extensive soft tissue hematoma, ectopic bone formation and compression of important structures and excessive bone resorption. These complications are all attributed to the excessive release of BMP-2. Therefore, the urgent problem to be solved at present is to find new carrier materials, so that BMPs can be actively and controlledly released through material degradation, reduce or eliminate the occurrence of complications, and expand the application field of BMPs in orthopedics. Although the Chinese patent document CN02114509.1 discloses a bone repair material with animal fibrin as a carrier, the material still has defects. It cannot protect BMPs from inhibition and degradation, and cannot effectively enhance the biological activity of BMPs.

【Content of invention】

One of the technical problems to be solved by the present invention is to provide an osteoinductive material, which can release BMPs in a small amount, efficiently and steadily, and effectively improve the biological activity of BMPs, and only need a small amount of BMPs to maintain the effective long-term release of BMPs .

The second technical problem to be solved by the present invention is to provide a preparation method of an osteoinductive material.

The third technical problem to be solved by the present invention is to provide the application of the osteoinductive material of the present invention in bone transplantation.

In order to solve the above technical problems, the present invention is realized through the following technical solutions:

In one aspect of the present invention, an osteoinductive material is provided, including an osteoinductive factor and a carrier, the osteoinductive factor comprising bone morphogenetic protein or basic fibroblast growth factor (basicfibroblast growth factor, bFGF), the The carrier is mainly composed of fibrinogen (fibrinogen), fibronectin (fibronectin), heparin, fibrin stabilization factor (fXIII factor), thrombin and calcium chloride to form a gel matrix through interaction. The binding is embedded in the gel matrix.

Preferably, the molar ratio of fibrinogen to fibronectin is 1-10:1; the molar ratio of fibronectin to heparin is 1-10:1.

Preferably, the osteoinductive factor is bone morphogenetic protein, the mass ratio of the protein to heparin is 1:1-50, and the bone morphogenetic protein is a natural bone morphogenetic protein extracted from animal bone or a Recombinant bone morphogenetic protein produced by engineering methods.

Preferably, the ratio between the fibrin stabilizing factor and the fibrinogen is: 0.3-1.2 IU of the fibrin stabilizing factor is corresponding to 1 mg of fibrinogen.

In another aspect of the present invention, a method for preparing an osteoinductive material is provided, comprising the steps of:

(1) taking osteoinductive factor comprising bone morphogenetic protein or basic fibroblast growth factor as component I, and preparing a solution containing fibrinogen, fibronectin, heparin and fibrin stabilizing factor as component II;

(2) preparing a solution containing thrombin and calcium chloride as component III;

(3) Add component II to component I to dissolve the inducer;

(4) Add component III to the mixed solution of components II and I to form an osteoinductive factor sustained release system.

Preferably, component I of the step (1) is bone morphogenetic protein.

Preferably, the concentration of the fibrinogen in the step (1) in the component II is 4-120 mg/ml.

Preferably, the concentration of thrombin in the step (2) in the component III is 40-800 IU/ml, and the concentration of the calcium chloride in the component III is 35-45umol/ml.

Preferably, the pH value of the components II and III is 6.8-9.

Preferably, after adding component II to component I in the step (3), place it in a water bath at 33° C. to 37° C. for 15 to 30 minutes.

Preferably, after adding component III to the mixed solution of components II and I in the step (4), put it into an incubator and incubate for 1-2 hours.

In another aspect of the present invention, the application of the osteoinductive material of the present invention in bone grafting is also provided.

The osteoinductive material of the present invention is a BMPs slow-release system, which is formed by fibrinogen and fibronectin solution under the action of thrombin and fibrin stabilizing factor to form a covalently cross-linked gel system; in the same reaction system BMPs are combined with heparin, and heparin is connected to the gel system due to its high affinity with fibronectin, so that BMPs are firmly embedded in the gel matrix to form a slow-release system. The osteoinductive material provided by the present invention is constructed by simulating the early temporary matrix of tissue repair. Fibrinogen, fibronectin and heparin are the main components of the early temporary matrix of tissue repair, and also represent the structure of the early temporary matrix of tissue repair. The fXIII factor is Necessary for the formation of this early temporary matrix of tissue repair; fibrinogen, fibronectin and heparin play an important role in the initial tissue repair process, heparin binds to BMPs, and heparin not only embeds BMPs in the matrix structure, but also protects BMPs are protected from inhibition and degradation, and the biological activity of BMPs is enhanced. The osteoinductive material composed of the sustained-release system of the present invention has the following advantages: 1) The BMPs can be actively sustained-release through material degradation mainly mediated by cells. 2) The sustained release of BMPs is trace, efficient, stable, without initial explosive release, and only needs a small amount of BMPs to maintain effective long-term release of BMPs. 3) Heparin has the function of protecting BMPs from degradation, and can also combine with BMPs inhibitory protein to prevent BMPs from being inactivated and effectively improve the biological activity of BMPs. 4) Due to the narrow concentration gradient formed by the slow release of BMPs, osteogenesis is limited. 5) The scaffold structure formed by fibrin and fibronectin is an early temporary matrix in the tissue repair process, which is conducive to the adhesion and invasion of cells and accelerates the osteogenesis process. 6) Compared with the rhBMP-2/ACS slow-release system, the release rate of BMPs in the slow-release system of the present invention can be adjusted by changing the constituent parameters of the slow-release system to meet the needs of different transplantation sites. 7) The clinical application of this slow-release system is very convenient, and it only needs 2-3 minutes to complete the one-step reaction. 8) The combination of this BMPs sustained-release system with other bone repair technologies (such as tissue-guided regeneration technology) also has great application value for the repair of bone defects.

This material is constructed by simulating the early temporary matrix of tissue repair. Fibrinogen, fibronectin and heparin are the main components of the early temporary matrix of tissue repair, and also represent the structure of the early temporary matrix of tissue repair. The fXIII factor is the formation of this early tissue repair Required for temporary substrate. Fibrinogen, fibronectin, and heparin play important roles in initiating tissue repair. Heparin combines with BMPs, heparin not only embeds BMPs in the matrix structure, but also protects BMPs from inhibition and degradation, and enhances the biological activity of BMPs

The osteoinductive material of the present invention not only overcomes the serious complication problem that may be caused by the rhBMP-2/ACS sustained release system, but also has better osteoconduction and osteoinduction capabilities. The osteoinductive material of the present invention has very good social benefits and market competitiveness because of its small amount of active component BMPs and reduced cost.

【Description of drawings】

Fig. 1 is a schematic diagram of a fibrin-fibronectin matrix containing a heparin-bound BMP slow-release system according to the present invention.

【Detailed ways】

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

The osteoinductive material of the present invention includes an osteoinductive factor and a carrier, the osteoinductive factor comprises bone morphogenetic protein or basic fibroblast growth factor, preferably, the osteoinductive factor of the present invention is a bone morphogenetic protein, and the bone morphogenetic Protein is natural bone morphogenetic protein extracted from animal bone or recombinant bone morphogenetic protein produced by genetic engineering method, the present invention preferably recombinant human bone morphogenetic protein-2 (rhBMP-2), and this genetic engineering product has high purity , high biological activity and stable character. The carrier is mainly composed of fibrinogen, fibronectin, heparin, fibrin stabilizing factor, thrombin and calcium chloride to form a gel matrix through interaction, and the osteoinductive factor is embedded in the gel matrix through the combination with heparin. in the gel matrix. The specific process of carrier formation is as follows: fibrinogen is cleaved into fibrin monomers under the action of thrombin, and the fibrin monomers are multimerized to form a gel network structure; on the other hand, factor fXIII is cleaved into fibrin monomers under the action of thrombin. Active fXIII factor (factor XIIIa), under the action of Ca ²⁺ , the active FXIII factor makes fibrin monomers covalently cross-link each other to form a stable network structure by virtue of its transglutaminase activity. The fibronectin molecule has many attachment sites for a variety of substrates, including fibrin, heparin, collagen, and integrins. The glutamine residue at the amino terminus of the fibronectin molecule is the glutamine transfer site for activated fXIII factor, through which the fibronectin molecule is covalently cross-linked with various other protein molecules, including fibrin, fiber proprotein as well as the fibronectin molecule itself. Heparin has a high affinity with fibronectin, and heparin can directly bind to BMP-2, so BMP-2 can be encapsulated in the carrier by heparin (see Figure 1), and the heparin helps to maintain BMP -2 activity, in the presence of heparin, the degradation of BMP-2 is inhibited, and its half-life can be extended 20 times in the culture medium.

In the present invention, the molar ratio of fibrinogen to fibronectin is 1 to 10:1; the molar ratio of fibronectin to heparin is 1 to 10:1; the molar ratio of bone morphogenetic protein to heparin The mass ratio is 1:1-50.

The ratio between the fibrin stabilizing factor and the fibrinogen is: 0.3-1.2 IU of the fibrin stabilizing factor is corresponding to 1 mg of fibrinogen.

The osteoinductive material of the present invention is constructed by simulating the formation of the early temporary matrix of tissue repair. Fibrinogen, fibronectin and heparin are the main components of the early temporary matrix of tissue repair, and also represent the structure of the early temporary matrix of tissue repair. The fXIII factor is the formation of this Necessary for the early provisional matrix of tissue repair. Fibrinogen, fibronectin, and heparin play important roles in initiating tissue repair. The three-dimensional scaffolding formed by fibrinogen and fibronectin provides a temporary matrix for cell adhesion and migration into the site of injury. This scaffold structure is remodeled by highly localized proteolytic degradation during cell invasion. There are receptors for plasminogen activator on the surface of the invading cells. When the receptor binds to plasminogen activator, plasminogen will be activated on the cell surface to produce plasmin, thereby degrading fibrin. This mode of action allows highly localized activation of plasminogen, leading to localized fiber hydrolysis rather than degradation of the entire matrix. The heparin combined with the scaffold structure embeds rhBMP-2 firmly in the matrix by means of electrostatic force. Heparanase enables the release of heparin-bound rhBMP-2 from the matrix. Studies have demonstrated that fibrin gel matrices modified to contain heparin binding sites can release heparin-bound rhBMP-2 in an active rather than passive manner. This active release is dependent on the cell-mediated action of plasmin and heparinase.

Example 1

The rhBMP-2 sustained-release system is temporarily prepared at the time of application, and is formed by mixing the following three components in sequence. Component I is rhBMP-2 lyophilized powder; Component II is a solution containing fibrinogen, fibronectin, heparin and factor XIII, pH 9.0, wherein the concentration of fibrinogen in Component II is 120mg/ ml, the molar ratio of fibrinogen to fibrinogen is 10:1, the molar ratio of fibrinogen to heparin is 10:1, in component II, every 1mg of fibrinogen corresponds to adding 1.2IU of Factor XIII; Component III is a solution containing thrombin and calcium chloride, pH9.0, the concentration of the thrombin in the component III is 800IU/ml, and the concentration of the _CaCl in the component III is 45umol /ml. During preparation, add component II to component I, and put it in a water bath at 37°C for 15 minutes to dissolve the rhBMP-2 lyophilized powder. The mass ratio of rhBMP-2 to heparin is 1:50, and then Add component III, shake quickly, put into an incubator, and incubate for 1-2 hours at 37° C. and 5% CO ₂ to form a gel. When used to treat single-segmental spinal degenerative lesions or bone defects, the gel matrix carrier containing rhBMP-2 is taken out and placed into an intervertebral fusion cage for implantation; for non-segmental bone defects, it can be directly The gel matrix carrier containing rhBMP-2 is placed in the defect; for segmental bone defects, the gel matrix carrier containing rhBMP-2 is first placed in the tissue-guided regeneration membrane, and then implanted in the bone defect, plus internal fixation.

Example 2

The rhBMP-2 sustained-release system is temporarily prepared at the time of application, and is formed by mixing the following three components in sequence. Component I is rhBMP-2 freeze-dried powder; Component II is a solution containing fibrinogen, fibronectin, heparin and factor XIII, pH 8.5, wherein the concentration of fibrinogen in Component II is 60mg/ ml, the molar ratio of fibrinogen to fibrinogen is 5: 1, the molar ratio of fibrinogen to heparin is 5: 1, in component II, every 1mg of fibrinogen corresponds to adding 0.65IU of Factor XIII; Component III is a solution containing thrombin and calcium chloride, pH8.6, the concentration of the thrombin in the component III is 450IU/ml, and the concentration of the _CaCl in the component III is 40umol /ml. During preparation, add component II to component I, and place it in a water bath at 35°C for 25 minutes to dissolve the rhBMP-2 lyophilized powder. The mass ratio of rhBMP-2 to heparin is 1:25, and then Add component III, shake quickly, put into an incubator, and incubate for 1-2 hours at 37° C. and 5% CO ₂ to form a gel. When used to treat single-segmental spinal degenerative lesions or bone defects, the gel matrix carrier containing rhBMP-2 is taken out and placed into an intervertebral fusion device for implantation; for non-segmental bone defects, the carrier containing The gel matrix carrier of rhBMP-2 is placed in the defect; for segmental bone defects, the gel matrix carrier containing rhBMP-2 is first placed in the tissue-guided regeneration membrane, and then implanted in the bone defect, plus internal fixation.

Example 3

The rhBMP-2 sustained-release system is temporarily prepared at the time of application, and is formed by mixing the following three components in sequence. Component I is rhBMP-2 lyophilized powder; Component II is a solution containing fibrinogen, fibronectin, heparin and factor XIII, pH6.8, wherein the concentration of fibrinogen in Component II is 4mg/ ml, the molar ratio of fibrinogen to fibrinogen is 1:1, the molar ratio of fibrinogen to heparin is 1:1, in component II, every 1mg of fibrinogen corresponds to adding 0.3IU of Factor XIII; Component III is a solution containing thrombin and calcium chloride, pH6.9, the concentration of the thrombin in the component III is 40IU/ml, and the concentration of the _CaCl in the component III is 35umol /ml. During preparation, add component II to component I, and dissolve in a water bath at 33°C for 30 minutes to fully dissolve the rhBMP-2 lyophilized powder. The mass ratio of rhBMP-2 to heparin is 1:1, and then Add component III, shake quickly, put into an incubator, and incubate for 1-2 hours at 37° C. and 5% CO ₂ to form a gel. When used to treat single-segmental spinal degenerative lesions or bone defects, the gel matrix carrier containing rhBMP-2 is taken out and placed into an intervertebral fusion device for implantation; for non-segmental bone defects, the carrier containing The gel matrix carrier of rhBMP-2 is placed in the defect; for segmental bone defects, the gel matrix carrier containing rhBMP-2 is first placed in the tissue-guided regeneration membrane, and then implanted in the bone defect, plus internal fixation.

Basic fibroblast growth factor (bFGF) can be used to treat soft tissue defects such as bone defects and skin according to the same steps as in the above-mentioned embodiments.

Example 4

The rhBMP-2 sustained-release system is temporarily prepared at the time of application, and is formed by mixing the following three components in sequence. Component I is rhBMP-2 freeze-dried powder; Component II is a solution containing fibrinogen, fibronectin, heparin and factor XIII, pH 8.0, wherein the concentration of fibrinogen in Component II is 90mg/ ml, the molar ratio of fibrinogen to fibrinogen is 5:1, the molar ratio of fibrinogen to heparin is 3:1, in component II, every 1mg of fibrinogen corresponds to adding 1.0IU of Factor XIII; Component III is a solution containing thrombin and calcium chloride, pH8.0, the concentration of the thrombin in the component III is 40IU/ml, and the concentration of the _CaCl in the component III is 40umol /ml. During preparation, add component II to component I, and put it in a water bath at 33°C for 30 minutes to dissolve the rhBMP-2 lyophilized powder. The mass ratio of rhBMP-2 to heparin is 1:3, and then Add component III, shake quickly, put into an incubator, and incubate for 1-2 hours at 37° C. and 5% CO ₂ to form a gel. When it is used to repair the critical defect of rat skull, the gel matrix carrier containing rhBMP-2 is taken out and implanted. At the same time, three control groups were also set up, which were group I blank control; group II fibrin glue plus rhBMP-2, lacking fibronectin and heparin, and the rest were the same as the experimental group; group III fibrin/fibronectin colloid plus rhBMP-2 2. Lack of heparin, the rest are the same as the experimental group. As a result of the experiment, the experimental group was better than the control group; in the control group, group III was the best, followed by group II. are statistically significant.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (13)

1. an osteoinductive material, comprising osteoinductive factor and carrier, is characterized in that, described osteoinductive factor comprises bone morphogenetic protein or basic fibroblast growth factor, and described carrier mainly consists of fibrinogen, fibronectin , heparin, fibrin stabilizing factor, thrombin and calcium chloride interact to form a gel matrix, and the osteoinductive factor is embedded in the gel matrix through the combination with heparin; wherein, the fibrinogen and The molar ratio of fibronectin is 1-10:1. 2. The osteoinductive material according to claim 1, characterized in that the molar ratio of the fibronectin to heparin is 1-10:1. 3. The osteoinductive material according to claim 1, wherein the osteoinductive factor is bone morphogenetic protein, and the mass ratio of the protein to heparin is 1:1-50. 4. The osteoinductive material according to claim 1 or 3, characterized in that, the bone morphogenetic protein is a natural bone morphogenetic protein extracted from animal bones or a recombinant bone morphogenetic protein produced by genetic engineering . 5 . The osteoinductive material according to claim 1 , wherein the ratio of the fibrin stabilizing factor to fibrinogen is: 0.3-1.2 IU of fibrin stabilizing factor is corresponding to 1 mg of fibrinogen. 6. A preparation method for an osteoinductive material, comprising the steps of: (1) The osteoinductive factor comprising bone morphogenetic protein or basic fibroblast growth factor is used as component I, and the solution containing fibrinogen, fibronectin, heparin and fibrin stabilizing factor is prepared as component II, wherein The molar ratio of the fibrinogen to the fibronectin is 1-10:1; (2) preparing a solution containing thrombin and calcium chloride as component III; (3) Add component II to component I to dissolve the inducer; (4) Add component III to the mixed solution of components II and I to form an osteoinductive factor sustained release system. 7. The preparation method according to claim 6, characterized in that, component I of the step (1) is bone morphogenetic protein. 8. The preparation method according to claim 6, characterized in that the concentration of the fibrinogen in the step (1) in the component II is 4-120 mg/ml. 9. preparation method according to claim 6, is characterized in that, the concentration of the thrombin in described step (2) in component III is 40～800IU/ml, and the concentration of described calcium chloride in component III The concentration is 35-45umol/ml. 10. The preparation method according to claim 6, characterized in that the pH values of the components II and III are 6.8-9. 11. The preparation method according to claim 6, characterized in that, after adding component II to component I in the step (3), place it in a water bath at 33° C. to 37° C. for 15 to 30 minutes. 12. The preparation method according to claim 6, characterized in that, in the step (4), after adding component III to the mixed solution of components II and I, put it into an incubator and incubate for 1-2 hours. 13. The application of the osteoinductive material according to claim 1 in bone grafting.

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