CN101249278B - Bone induction material and preparation method and application thereof - Google Patents
Bone induction material and preparation method and application thereof Download PDFInfo
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- CN101249278B CN101249278B CN2008100658631A CN200810065863A CN101249278B CN 101249278 B CN101249278 B CN 101249278B CN 2008100658631 A CN2008100658631 A CN 2008100658631A CN 200810065863 A CN200810065863 A CN 200810065863A CN 101249278 B CN101249278 B CN 101249278B
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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
[technical field]
The present invention relates to osseous tissue renovating material, relate in particular to a kind of bone induction material and its production and application.
[background technology]
Bone is to be only second to the most common the passing through of blood to transplant the tissue of repairing.In the U.S., the 500000 routine bone transplant operations of surpassing there is every year.The whole world is annual to be that the repairing bone defect bone transplant operation of carrying out is up to 2,200,000 examples in orthopaedics, neurosurgery and dentistry.Account for half of bone transplant operation sum in the application of spinal surgery.Along with the aging of population, need the surge of the spinal column degeneration case of treatment, the application that bone is implanted in spinal surgery increases sharply.It is to be used for treating the surgical method that bone above the body healing ability is damaged and carry out bone fusion between vertebral body that bone is transplanted.At spinal surgery, all face the problem of keeping spinal stabilization behind the spinal operation; Between operation sections vertebral body or behind the spinal column between the transverse process of the outside implantable bone integrating materials be the effective ways that recover and keep the spinal column long-time stability.Invasive lumbar fusion device (interbody fusion cage) and bone grafting material use in conjunction are the recommendation art formulas of treatment single-unit section spinal column degeneration.Invasive lumbar fusion device is after intervertebral disc is removed, and is placed on the hollow structure that comes stabilizing spine between adjacent vertebral bodies and recover height between vertebral body.Bone grafting material just is placed on the intermediary space of Invasive lumbar fusion device, connects adjacent vertebral bodies by bony union, keeps the long-term stability of spinal column.
Present employed bone grafting material has from body bone, homogeneous allogenic bone and synthetic bone substitute.From the body bone is the goldstandard of bone graft, but it is limited to originate, and can cause new damage to body when obtaining; The homogeneous allogenic bone wide material sources, but immunologic rejection and risk of disease transmission are arranged.Above-mentioned these deficiencies have limited them in clinical extensive use.In order to satisfy clinical demand, constantly there is new synthetic bone substitution material to obtain development and utilization.This comprises natural material, polymer, bioactive ceramics and several short skeletal growth factor.Short skeletal growth factor is induced because of it is strong or the ability of promote osteogenesis just is being subjected to extensive studies and application.Wherein, (bone morphogenetic proteins BMPs) is the osteogenetic somatomedin of the most strong promotion of at present known unique energy induced dry-cell Osteoblast Differentiation to bone morphogenetic protein.The BMPs aqueous solution is expelled to the interior skeletonization site of body and can be removed by body very soon, does not reach effective skeletonization.BMPs has only and carrier composition slow-released system, maintains localized sustained and effectively discharges, and just can obtain the skeletonization effect.(absorbable collagen sponge, ACS) slow-released system of Zu Chenging is induced packing material to be used for the lumbar vertebra anterior approach by U.S. food and drug administration (FDA) approval as the bone of Invasive lumbar fusion device to the adsorbable type i collagen sponge of rhBMP-2 (rhBMP-2) and cattle.The BMP-2/ACS slow-released system is developed to series of products by U.S. pivot Fa Mo company, introduces to the market with the INFUSE trade name, is used from the preceding road of lumbar vertebra with Invasive lumbar fusion device (LT-cage) and merges.A large amount of secular clinical researches show that the rhBMP-2/ACS slow-released system is used for the preceding road of lumbar vertebra and merges significantly better than autogenous bone graft.This shows that BMPs combines with appropriate carrier to be expected to replace becomes new bone from the body bone and transplants goldstandard.Yet also there is tangible deficiency in the rhBMP-2/ACS slow-released system.This slow-released system is to discharge by passive simple diffusion in vivo, exists initial burst to discharge; This causes the controllable sustained-release effect not remarkable, the using in a large number and wasting of active factors, and the skeletonization scope should not be limited to.Because using at lumbar vertebra, the relation of anatomical position, rhBMP-2/ACS slow-released system do not have tangible complication; But the incidence rate of complication is quite high when other positions, shows as extensive soft tissue hematoma, dystopy skeletonization compressing important structure and bone taken in excess.These complication are all owing to the excessive release of BMP-2.Therefore, current urgent problem is to seek new carrier material, makes BMPs pass through material degradation and produces active controllable release, reduces or eliminate the generation of complication, opens up the application of wide BMPs at orthopaedics.Though it is the bone renovating material of carrier with the animal fibrin that Chinese patent literature CN02114509.1 discloses a kind of, still there is defective in this material, and it can not protect BMPs to avoid suppressing and degraded, can not effectively promote the biological activity of BMPs.
[summary of the invention]
One of the technical problem to be solved in the present invention provides a kind of bone induction material, can trace, slow release BMPs efficiently and reposefully, and effectively improve the biological activity of BMPs, and only need a small amount of BMPs, just can keep the effectively long-term release of BMPs.
Two of the technical problem to be solved in the present invention provides a kind of preparation method of bone induction material.
Three of the technical problem to be solved in the present invention provides the application of bone induction material of the present invention in bone is transplanted.
In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of bone induction material is provided, comprise bone-inducing factor and carrier, described bone-inducing factor comprises bone morphogenetic protein or basic fibroblast growth factor (basicfibroblast growth factor, bFGF), described carrier is mainly by Fibrinogen (fibrinogen), Fn Fiberonectin (fibronectin), heparin, fibrin stabilizing factor (the fXIII factor), thrombin and calcium chloride form gel-type vehicle through interacting, and described bone-inducing factor is by being embedded in this gel-type vehicle with combining of heparin.
Preferably, the mol ratio of described Fibrinogen and Fn Fiberonectin is 1～10: 1; The mol ratio of described Fn Fiberonectin and heparin is 1～10: 1.
Preferably, described bone-inducing factor is a bone morphogenetic protein, the mass ratio of this albumen and heparin is 1: 1～50, and described bone morphogenetic protein is nature bone morphogenetic proteins that extracts from animal bone or the reorganization bone morphogenetic protein of producing with gene engineering method.
Preferably, described fibrin stabilizing factor with fibrinogenic proportionate relationship is: the fibrin stabilizing factor of the corresponding 0.3～1.2IU of every 1mg Fibrinogen.
In another aspect of this invention, provide a kind of preparation method of bone induction material, comprised the steps:
(1) bone-inducing factor that will comprise bone morphogenetic protein or basic fibroblast growth factor is as component I, and preparation contains the solution of Fibrinogen, Fn Fiberonectin, heparin and fibrin stabilizing factor as component I I;
(2) preparation contains the solution of thrombin and calcium chloride as component III;
(3) component I I is added component I, with the dissolving inducible factor;
(4) component III is added the mixed solution of component I I and I, to form the bone-inducing factor slow-released system.
Preferably, the component I of described step (1) is a bone morphogenetic protein.
Preferably, the concentration of Fibrinogen in component I I of described step (1) is 4～120mg/ml.
Preferably, the concentration of thrombin in component III of described step (2) is 40～800IU/ml, and the concentration of described calcium chloride in component III is 35～45umol/ml.
Preferably, the pH value of described component I I and III is 6.8～9.
Preferably, in the described step (3) component I I is added after the component I, in 33 ℃～37 ℃ water-baths, placed 15～30 minutes.
Preferably, in the described step (4) component III is added after the mixed solution of component I I and I, put into incubator and hatched 1～2 hour.
In another aspect of this invention, also provide the application of bone induction material of the present invention in bone is transplanted.
Bone induction material of the present invention is the BMPs slow-released system, and this slow-released system forms covalent cross-linking gel rubber system each other by Fibrinogen and Fn Fiberonectin solution under the effect of thrombin and fibrin stabilizing factor; BMPs and heparin combine in same reaction system, and heparin is because of having high-affinity to be connected to this gel rubber system with Fn Fiberonectin, and BMPs firmly is embedded in and forms slow-released system in the gel-type vehicle like this.Bone induction material provided by the invention is the early stage interim substrate of simulated tissue reparation and making up, Fibrinogen, Fn Fiberonectin and heparin are the main components of the early stage interim substrate of tissue repair, also represent the structure of the early stage interim substrate of tissue repair, the fXIII factor is that the early stage interim substrate of this tissue repair of formation is necessary; Fibrinogen, Fn Fiberonectin and heparin play a significant role in initial process of tissue reparation, heparin and BMPs combination, and heparin not only is embedded in BMPs in the matrix structure, also protects BMPs to avoid suppressing and degraded, promotes the biological activity of BMPs.The bone induction material that the present invention is made up of slow-released system has following advantage: 1) make BMPs pass through mainly to produce initiatively slow release by cell-mediated material degradation.2) the slow release trace of BMPs, efficient, steady, no initial burst release only needs BMPs on a small quantity, just can keep effective long-term release of BMPs.3) heparin has the effect that protection BMPs avoids degrading, and can also combine with the BMPs Profilin, stops BMPs to be inactivated, and effectively improves the biological activity of BMPs.4) because BMPs slowly discharges formed Concentraton gradient narrow range, the skeletonization limitation.5) the formed supporting structure of fibrin and Fn Fiberonectin is the early stage interim substrate of process of tissue reparation, and this substrate helps the adhesion and the intrusion of cell, quickens osteogenetic process.6) compare with the rhBMP-2/ACS slow-released system, the release rate of the BMPs of slow-released system of the present invention can be regulated by the formation parameter that changes slow-released system, to satisfy the needs of different transplanted sites.7) clinical practice of this slow-released system is very convenient, only needs 2-3 minute single step reaction just can finish.8) this BMPs slow-releasing system combines with other bone recovery techniques (for example guided tissue regeneration's technology), and damaged reparation also has huge using value for bone.
This material is that the early stage interim substrate of simulated tissue reparation makes up, Fibrinogen, Fn Fiberonectin and heparin are the main components of the early stage interim substrate of tissue repair, also represent the structure of the early stage interim substrate of tissue repair, the fXIII factor is that the early stage interim substrate of this tissue repair of formation is necessary.Fibrinogen, Fn Fiberonectin and heparin play a significant role in initial process of tissue reparation.Heparin and BMPs combination, heparin not only is embedded in BMPs in the matrix structure, also protects BMPs to avoid suppressing and degraded, promotes the biological activity of BMPs
Bone induction material of the present invention has not only overcome the severe complication problem that the rhBMP-2/ACS slow-released system may cause, and has better bone conduction and bone inducibility.Bone induction material of the present invention is few because of its active component BMPs consumption, and cost reduces, and has the good social benefit and the market competitiveness.
[description of drawings]
The present invention of being shown in Figure 1 contains the fibrin-Fn Fiberonectin substrate sketch map of heparin in conjunction with the BMP slow-released system.
[specific embodiment]
The present invention is further detailed explanation below in conjunction with drawings and Examples.
Bone induction material of the present invention comprises bone-inducing factor and carrier, described bone-inducing factor comprises bone morphogenetic protein or basic fibroblast growth factor, preferably, bone-inducing factor of the present invention is a bone morphogenetic protein, this bone morphogenetic protein is nature bone morphogenetic proteins that extracts from animal bone or the reorganization bone morphogenetic protein of producing with gene engineering method, the preferred rhBMP-2 of the present invention (rhBMP-2), this gene engineering product has the stable characteristics of high-purity, high bioactivity and moral character.Described carrier mainly forms gel-type vehicle by Fibrinogen, Fn Fiberonectin, heparin, fibrin stabilizing factor, thrombin and calcium chloride through interacting, and described bone-inducing factor is by being embedded in this gel-type vehicle with combining of heparin.The detailed process that carrier forms is as follows: Fibrinogen is cracked into fibrin monomer under the effect of thrombin, and the fibrin monomer multimerization forms gel network structure; The fXIII factor is cracked under the effect of thrombin and has the active fXIII factor (factor XIIIa) on the other hand, and the active FXIII factor is at Ca
2+Effect under, rely on the activity of its transglutaminase, make fibrin monomer each other covalent cross-linking form the stabilizing network structure.The Fn Fiberonectin molecule has many various substrate attachment sites, and substrate comprises fibrin, heparin, collagen and integration element etc.In the N-terminal glutamine residue of Fn Fiberonectin molecule is the glutamine transfer site of the activation fXIII factor, mutually crosslinked with various other protein molecular covalency the Fn Fiberonectin molecule by this site, this comprises fibrin, Fibrinogen and Fn Fiberonectin molecule self.Heparin and Fn Fiberonectin have high affinity, and heparin can directly combine with BMP-2, therefore BMP-2 can be wrapped in the carrier by heparin and (see accompanying drawing 1), and described heparin helps to keep the activity of BMP-2, under the heparin existence condition, the degraded of BMP-2 is suppressed, and its half-life can prolong 20 times in culture fluid.
In the present invention, the mol ratio of described Fibrinogen and Fn Fiberonectin is 1～10: 1; The mol ratio of described Fn Fiberonectin and heparin is 1～10: 1; The mass ratio of described bone morphogenetic protein and heparin is 1: 1～50.
Described fibrin stabilizing factor with fibrinogenic proportionate relationship is: the fibrin stabilizing factor of the corresponding 0.3～1.2IU of every 1mg Fibrinogen.
The early stage interim substrate of bone induction material simulated tissue of the present invention reparation forms and makes up, Fibrinogen, Fn Fiberonectin and heparin are the main components of the early stage interim substrate of tissue repair, also represent the structure of the early stage interim substrate of tissue repair, the fXIII factor is that the early stage interim substrate of this tissue repair of formation is necessary.Fibrinogen, Fn Fiberonectin and heparin play a significant role in initial process of tissue reparation.The formed three-dimensional rack structure of Fibrinogen and Fn Fiberonectin is advanced injury site for cell adhesion and migration provides temporary transient substrate.This supporting structure is reinvented by the Proteolytic enzyme degraded of highly limiting between stage of invasion at cell.There is the receptor of activator of plasminogen on the surface of invading cell, when receptor combines with activator of plasminogen, can produce fibrinolysin at this place's cell surface plasminogen activation, thus fibrin degradation.This model of action can cause the hydrolysis of limitation fiber to the activation of plasminogen height localization, rather than the monoblock substrate degradation.The bonded heparin of supporting structure institute firmly is embedded in rhBMP-2 in the middle of the substrate by electrostatic force.Heparinase can make heparin-bounding rhBMP-2 discharge from substrate.Research has proved the modified heparin binding site that contains of fibrin gel substrate, can discharge heparin-bounding rhBMP-2 with active mode rather than passive mode.This active discharges the effect that depends on cell-mediated fibrinolysin and heparinase.
Embodiment 1
The rhBMP-2 slow-released system prepares when using temporarily, is mixed successively by following three kinds of components.Component I is the rhBMP-2 lyophilized powder; Component I I is the solution that contains Fibrinogen, Fn Fiberonectin, heparin and the XIII factor, PH9.0, wherein, the concentration of Fibrinogen in component I I is 120mg/ml, the mol ratio of described Fibrinogen and Fn Fiberonectin is 10: 1, the mol ratio of described Fn Fiberonectin and heparin is 10: 1, in component I I, and the corresponding XIII factor that adds 1.2IU of every 1mg Fibrinogen; Component III is the solution that contains thrombin and calcium chloride, PH9.0, and the concentration of described thrombin in component III is 800IU/ml, described CaCl
2Concentration in component III is 45umol/ml.In the preparation, component I I is added component I, and place 37 ℃ of water-bath dissolvings 15 minutes, with abundant dissolving rhBMP-2 lyophilized powder, the mass ratio of this rhBMP-2 and heparin is 1: 50, and then adds component III, shake up fast, put into incubator, at 37 ℃, 5%CO
2Condition under, hatched 1～2 hour, to form gel.When being used for the treatment of single-unit section spinal column degeneration or bone when damaged, this gel-type vehicle carrier that comprises rhBMP-2 taken out put in the middle of the Invasive lumbar fusion device, implant; Damaged for non-sections bone, can directly the gel-type vehicle carrier that comprises rhBMP-2 be put into damaged place; Damaged for the sections bone, the gel-type vehicle carrier that comprises rhBMP-2 is put into earlier in the middle of guided tissue regeneration's film, and the damaged place of implantable bone adds internal fixation again.
Embodiment 2
The rhBMP-2 slow-released system prepares when using temporarily, is mixed successively by following three kinds of components.Component I is the rhBMP-2 lyophilized powder; Component I I is the solution that contains Fibrinogen, Fn Fiberonectin, heparin and the XIII factor, PH8.5, wherein, the concentration of Fibrinogen in component I I is 60mg/ml, the mol ratio of described Fibrinogen and Fn Fiberonectin is 5: 1, the mol ratio of described Fn Fiberonectin and heparin is 5: 1, in component I I, and the corresponding XIII factor that adds 0.65IU of every 1mg Fibrinogen; Component III is the solution that contains thrombin and calcium chloride, PH8.6, and the concentration of described thrombin in component III is 450IU/ml, described CaCl
2Concentration in component III is 40umol/ml.In the preparation, component I I is added component I, and place 35 ℃ of water-bath dissolvings 25 minutes, with abundant dissolving rhBMP-2 lyophilized powder, the mass ratio of this rhBMP-2 and heparin is 1: 25, and then adds component III, shake up fast, put into incubator, at 37 ℃, 5%CO
2Condition under, hatched 1～2 hour, to form gel.When being used for the treatment of single-unit section spinal column degeneration or bone when damaged, this gel-type vehicle carrier that comprises rhBMP-2 taken out put into Invasive lumbar fusion device, implant; Damaged for non-sections bone, can directly the gel-type vehicle carrier that comprises rhBMP-2 be put into damaged place; Damaged for the sections bone, the gel-type vehicle carrier that comprises rhBMP-2 is put into earlier in the middle of guided tissue regeneration's film, and the damaged place of implantable bone adds internal fixation again.
Embodiment 3
The rhBMP-2 slow-released system prepares when using temporarily, is mixed successively by following three kinds of components.Component I is the rhBMP-2 lyophilized powder; Component I I is the solution that contains Fibrinogen, Fn Fiberonectin, heparin and the XIII factor, PH6.8, wherein, the concentration of Fibrinogen in component I I is 4mg/ml, the mol ratio of described Fibrinogen and Fn Fiberonectin is 1: 1, the mol ratio of described Fn Fiberonectin and heparin is 1: 1, in component I I, and the corresponding XIII factor that adds 0.3IU of every 1mg Fibrinogen; Component III is the solution that contains thrombin and calcium chloride, PH6.9, and the concentration of described thrombin in component III is 40IU/ml, described CaCl
2Concentration in component III is 35umol/ml.In the preparation, component I I is added component I, and place 33 ℃ of water-bath dissolvings 30 minutes, with abundant dissolving rhBMP-2 lyophilized powder, the mass ratio of this rhBMP-2 and heparin is 1: 1, and then adds component III, shakes up fast, puts into incubator, at 37 ℃, 5%CO
2Condition under, hatched 1～2 hour, to form gel.When being used for the treatment of single-unit section spinal column degeneration or bone when damaged, this gel-type vehicle carrier that comprises rhBMP-2 taken out put into Invasive lumbar fusion device, implant; Damaged for non-sections bone, can directly the gel-type vehicle carrier that comprises rhBMP-2 be put into damaged place; Damaged for the sections bone, the gel-type vehicle carrier that comprises rhBMP-2 is put into earlier in the middle of guided tissue regeneration's film, and the damaged place of implantable bone adds internal fixation again.
Basic fibroblast growth factor (bFGF) can be used for the treatment of soft tissue defects such as bone is damaged, skin by the same steps as of the foregoing description.
Embodiment 4
The rhBMP-2 slow-released system prepares when using temporarily, is mixed successively by following three kinds of components.Component I is the rhBMP-2 lyophilized powder; Component I I is the solution that contains Fibrinogen, Fn Fiberonectin, heparin and the XIII factor, PH8.0, wherein, the concentration of Fibrinogen in component I I is 90mg/ml, the mol ratio of described Fibrinogen and Fn Fiberonectin is 5: 1, the mol ratio of described Fn Fiberonectin and heparin is 3: 1, in component I I, and the corresponding XIII factor that adds 1.0IU of every 1mg Fibrinogen; Component III is the solution that contains thrombin and calcium chloride, PH8.0, and the concentration of described thrombin in component III is 40IU/ml, described CaCl
2Concentration in component III is 40umol/ml.In the preparation, component I I is added component I, and place 33 ℃ of water-bath dissolvings 30 minutes, with abundant dissolving rhBMP-2 lyophilized powder, the mass ratio of this rhBMP-2 and heparin is 1: 3, and then adds component III, shakes up fast, puts into incubator, at 37 ℃, 5%CO
2Condition under, hatched 1～2 hour, to form gel.Critical when damaged when being used to repair the rat skull, this gel-type vehicle carrier that comprises rhBMP-2 taken out implant.Also setting up three matched groups simultaneously, is respectively group I blank; Group II Fibrin Glue adds rhBMP-2, lacks Fn Fiberonectin and heparin, and all the other are identical with experimental group; Group III fibrin/Fn Fiberonectin colloid adds rhBMP-2, lacks heparin, and all the other are identical with experimental group.Experimental result, experimental group is better than matched group; In the matched group, III is best for group, and group II takes second place.All has statistical significance.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (13)
1. bone induction material, comprise bone-inducing factor and carrier, it is characterized in that, described bone-inducing factor comprises bone morphogenetic protein or basic fibroblast growth factor, described carrier mainly forms gel-type vehicle by Fibrinogen, Fn Fiberonectin, heparin, fibrin stabilizing factor, thrombin and calcium chloride through interacting, and described bone-inducing factor is by being embedded in this gel-type vehicle with combining of heparin; Wherein, the mol ratio of described Fibrinogen and Fn Fiberonectin is 1～10: 1.
2. bone induction material according to claim 1 is characterized in that, the mol ratio of described Fn Fiberonectin and heparin is 1～10: 1.
3. bone induction material according to claim 1 is characterized in that, described bone-inducing factor is a bone morphogenetic protein, and the mass ratio of this albumen and heparin is 1: 1～50.
4. according to claim 1 or 3 described bone induction materials, it is characterized in that described bone morphogenetic protein is nature bone morphogenetic proteins that extracts or the reorganization bone morphogenetic protein of producing with gene engineering method from animal bone.
5. bone induction material according to claim 1 is characterized in that, described fibrin stabilizing factor with fibrinogenic proportionate relationship is: the fibrin stabilizing factor of the corresponding 0.3～1.2IU of every 1mg Fibrinogen.
6. the preparation method of a bone induction material is characterized in that, comprises the steps:
(1) will comprise the bone-inducing factor of bone morphogenetic protein or basic fibroblast growth factor as component I, the solution that preparation contains Fibrinogen, Fn Fiberonectin, heparin and fibrin stabilizing factor is as component I I, and the mol ratio of wherein said Fibrinogen and Fn Fiberonectin is 1～10: 1;
(2) preparation contains the solution of thrombin and calcium chloride as component III;
(3) component I I is added component I, with the dissolving inducible factor;
(4) component III is added the mixed solution of component I I and I, to form the bone-inducing factor slow-released system.
7. preparation method according to claim 6 is characterized in that, the component I of described step (1) is a bone morphogenetic protein.
8. preparation method according to claim 6 is characterized in that, the concentration of Fibrinogen in component I I of described step (1) is 4～120mg/ml.
9. preparation method according to claim 6 is characterized in that, the concentration of thrombin in component III of described step (2) is 40～800IU/ml, and the concentration of described calcium chloride in component III is 35～45umol/ml.
10. preparation method according to claim 6 is characterized in that, the pH value of described component I I and III is 6.8～9.
11. preparation method according to claim 6 is characterized in that, in the described step (3) component I I is added after the component I, places 15～30 minutes in 33 ℃～37 ℃ water-baths.
12. preparation method according to claim 6 is characterized in that, in the described step (4) component III is added after the mixed solution of component I I and I, puts into incubator and hatches 1～2 hour.
13. the application of the described bone induction material of claim 1 in bone is transplanted.
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| CN103933619B (en) * | 2014-04-29 | 2017-02-15 | 深圳清华大学研究院 | Nerve repairing material and preparation method thereof |
| PL3697459T3 (en) * | 2017-10-19 | 2023-03-06 | Genera Istrazivanja D.O.O. | Autologous bone graft substitute |
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