CN104474591A - Bioactive degradable coral hydroxyapatite artificial bone - Google Patents
Bioactive degradable coral hydroxyapatite artificial bone Download PDFInfo
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- CN104474591A CN104474591A CN201410843797.1A CN201410843797A CN104474591A CN 104474591 A CN104474591 A CN 104474591A CN 201410843797 A CN201410843797 A CN 201410843797A CN 104474591 A CN104474591 A CN 104474591A
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- releasing agent
- slow releasing
- artificial bone
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Links
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 227
- 229910052588 hydroxylapatite Inorganic materials 0.000 title claims abstract description 97
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 title claims abstract description 97
- 235000014653 Carica parviflora Nutrition 0.000 title claims abstract description 87
- 241000243321 Cnidaria Species 0.000 title claims abstract description 87
- 230000000975 bioactive effect Effects 0.000 title abstract 2
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- 238000002360 preparation method Methods 0.000 claims abstract description 19
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- 108010035532 Collagen Proteins 0.000 claims abstract description 14
- 229920001436 collagen Polymers 0.000 claims abstract description 14
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 9
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 9
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 9
- 102000016359 Fibronectins Human genes 0.000 claims abstract description 8
- 108010067306 Fibronectins Proteins 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 77
- 241001450685 Corallium japonicum Species 0.000 claims description 47
- 108090000695 Cytokines Proteins 0.000 claims description 41
- 102000004127 Cytokines Human genes 0.000 claims description 41
- 239000007864 aqueous solution Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000008367 deionised water Substances 0.000 claims description 19
- 229910021641 deionized water Inorganic materials 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 16
- 238000004140 cleaning Methods 0.000 claims description 14
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 claims description 12
- 102000045896 human BMP2 Human genes 0.000 claims description 12
- 239000011148 porous material Substances 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 8
- 238000005286 illumination Methods 0.000 claims description 8
- 229910001410 inorganic ion Inorganic materials 0.000 claims description 8
- 238000005498 polishing Methods 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 4
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- 239000008240 homogeneous mixture Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention relates to the technical field of medical biological materials, in particular to a bioactive degradable coral hydroxyapatite artificial bone and a preparation method thereof. The artificial bone comprises a bracket, wherein bone-inducing active cell factors and controlled release formulations are attached onto the bracket; the bracket comprises a bracket body; the bracket body is coral; a hydroxyapatite layer coats the surface of the bracket body; the bone-inducing active cell factors are made of recombinant human bone morphogenetic protein-2 mature peptide; the controlled release formulations are selected from collagen, fibronectin, hyaluronic acid and the like; the bracket is granular or cylindrical; the aperture range of micropores is 77-180 microns; the porosity of the micropores is 8-11%. According to verification results of the National Institute for the Control of Pharmaceutical and Biological Products (verification report numbers comprise QZ200701428 and QZ200804480) and the National Institutes for Food and Drug control (verification report number is QZ201206312), the artificial bone is excellent bone graft substitute.
Description
Technical field
The present invention relates to medical biomaterial technical field, particularly relate to a kind of biological degradable coral hydroxyapatite artificial bone and preparation method thereof.
Background technology
The patient of Cranial defect occupies very high ratio in orthopedics patient, and this kind of patient by human body self-recovery mechanism repair deficiency, therefore cannot must carry out bone collection treatment.
For bone collection treatment material referred to as artificial bone, artificial bone needs suitable size, suitable porosity and aperture, and has suitable material and function.
Desirable bone grafting material, needs to possess bone conduction and bone-inducting active simultaneously.Corallium Japonicum Kishinouye has the pore structure similar to human bone with the hydroxyapatite material derived by Corallium Japonicum Kishinouye, the filling of Cranial defect is widely used in as timbering material, because it has good 3-D solid structure and biomechanical property, therefore outstanding bone conduction is shown active, but the inducibility of area of new bone is not enough, osteogenetic process is slow, especially cannot obtain satisfied curative effect for large intercept Cranial defect and large area Cranial defect.In addition, Corallium Japonicum Kishinouye main component CaCO
3, implants and be degraded very soon, be not enough to maintain whole osteogenetic process, and hydroxyapatite degradation in vivo speed is extremely slow, affects substituting of autologous tissue.BMP-2 is one of cytokine in human body with induction bone formation ability the most by force, the use of BMP-2 can accelerating union of bone fracture and new bone formation process, but, BMP-2 is small protein, easy diffusion, concentration of can not remaining valid in the region needing skeletonization, continues to play bone inductive effect, although therefore cytokine has good bone-inducing activity, be used alone and still cannot play positive effect in the repair process of Cranial defect.
Therefore, for tissue engineering artificial bone, the use of single key element has been difficult to meet clinically to the demand of bone graft substitute, and current investigation and application has progressively turned to the compound use of many key elements, to build more effectively more reasonably artificial bone.
Summary of the invention
In order to the degradability solving existing artificial bone is undesirable, the defect that bone-inducting active is poor, the invention provides a kind of biological degradable coral hydroxyapatite artificial bone and preparation method thereof.
In order to solve the problems of the technologies described above, the present invention adopts following technical proposals:
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is selected from the combination of a kind of in collagen, fibronectin, hyaluronic acid, chitin, polyvinylpyrrolidone or at least two kinds.
Further, described support is irregular 3-D solid structure, and the scope of length, width and height is 2-20mm, and the pore diameter range of described micropore is 77-200 μm, and the porosity of described micropore is 8%-11%.
Further, described artificial bone is grain type or column type.
Further, grain type artificial bone: diameter is 2-6mm.
Further, column type artificial bone: body diameter is 10 ± 0.5mm; Cylinder is high is 20 ± 1.0mm.
Further, the pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
Further, the aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。(see sequence 1)
Further, the concentration of described bone-inducing activity cytokine is 0.1-1mg/cm
3, the concentration of described slow releasing agent is 0.2-2%.
The concentration of described bone-inducing activity cytokine is 0.1-1mg/cm
3, refer to that the support of every cubic centimetre of artificial bone is interior containing 0.1-1mg bone-inducing activity cytokine.The concentration of described slow releasing agent is 0.2-2%, refers to that the support of every 100 cubic centimetres of artificial bones is interior containing 0.2-2g slow releasing agent.
Further, the concentration of described bone-inducing activity cytokine is 0.6-1mg/cm
3, the concentration of described slow releasing agent is 0.2%.
Further, the concentration of described bone-inducing activity cytokine is 0.6-1mg/cm
3, the concentration of described slow releasing agent is 1-2%.
Further, described slow releasing agent is collagen, fibronectin, hyaluronic acid and chitinous combination, and the mass ratio of aforementioned four kinds of slow releasing agents is 1:1:1:1.
The present invention also provides the preparation method of above-mentioned degradable coral hydroxyapatite artificial bone, and described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; Cleaning; Baking oven is put at 50-100 DEG C of dry 3-4 hour after cleaning up; Then, by Corallium Japonicum Kishinouye and (NH
4)
2hPO
4aqueous solution together put into autoclave, (NH
4)
2hPO
4the mass concentration of aqueous solution is 0.05-1mol/L, (NH
4)
2hPO
4(NH in solution
4)
2hPO
4with Corallium Japonicum Kishinouye (CaCO
3) mol ratio be 1:10-15; Again to (NH
4)
2hPO
4potassium dihydrogen phosphate is added, often liter of (NH in solution
4)
2hPO
4potassium dihydrogen phosphate 1mg is added in solution; React 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Use gamma-rays illumination-based disinfection again, obtain degradable coral hydroxyapatite support;
(2) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are mixed with the aqueous solution of mixing homogeneously.
(3) in surface recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent being attached to degradable coral hydroxyapatite support and hole.
Further, the preparation method of described degradable coral hydroxyapatite artificial bone, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; Cleaning; Baking oven is put at 50-100 DEG C of dry 3-4 hour after cleaning up; Then, by Corallium Japonicum Kishinouye and (NH
4)
2hPO
4aqueous solution together put into autoclave, (NH
4)
2hPO
4the mass concentration of aqueous solution is 0.05-1mol/L, (NH
4)
2hPO
4(NH in solution
4)
2hPO
4with Corallium Japonicum Kishinouye (CaCO
3) mol ratio be 1:10-1:15; Again to (NH
4)
2hPO
4potassium dihydrogen phosphate is added, often liter of (NH in solution
4)
2hPO
4potassium dihydrogen phosphate 1mg is added in solution; React 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Use gamma-rays illumination-based disinfection again, obtain degradable coral hydroxyapatite support, finally by dry for degradable coral hydroxyapatite support subpackage cillin bottle;
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the aqueous solution of concentration 0.1-1mg/ml, the slow releasing agent accounting for aqueous solution gross weight 0.2%-2% is added in aqueous solution, under low temperature, low speed is stirred to mixing fully, obtains the homogeneous mixture solotion of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite support
Be added to by the mixed solution that step (2) obtains in degradable coral hydroxyapatite support that step (1) obtains, addition is 0.1-1mg/cm
3(that is: every cubic centimetre of support adds the mixed solution containing 0.1-1mg recombinant human bone morphorgenetic protein-2 mature peptide), low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent fully enter in the hole of degradable coral hydroxyapatite support, less than-30 DEG C evacuation, lyophilization, obtain described artificial bone, artificial bone is sealed, preserves for a long time for-20 DEG C.
In step (1), the main component of natural coral is calcium carbonate (CaCO
3), warp and (NH
4)
2hPO
4reaction can make surface portion be converted into hydroxyapatite.
Further, the cleaning process in step (1) first uses ultrasonic cleaning, rear running water 20 minutes, to remove organic principle in Surface of Coral and internal void and Corallium Japonicum Kishinouye chip.
Further, in the preparation method of described degradable coral hydroxyapatite artificial bone, the pressure in described step (1) in reactor is: 4-9MPa, temperature is: 270-280 DEG C.
Further, in the preparation method of described degradable coral hydroxyapatite artificial bone, Corallium Japonicum Kishinouye and (NH in described step (1)
4)
2hPO
4ratio be: 10:1, the pressure in reactor is: 7MP, and temperature is: 280 DEG C.
Further, in described step (2), cryogenic temperature is 2 DEG C-6 DEG C, and stirring at low speed rotating speed is 50-150 rev/min.
Further, in described step (3), cryogenic temperature is 1-5 DEG C, and negative pressure is-0.01 to-0.05MPa.
Compared with prior art, biological degradable coral hydroxyapatite artificial bone provided by the present invention has following advantage:
1, possess the active and bone-inducting active of good bone conduction simultaneously, namely have good biomechanics characteristic, with pore structure like autologous bone photo and good histocompatibility, the newborn osteoplastic ability of induction of BMP-2 can be given full play to again.Having good bone defect healing ability, is a kind of desirable bone graft substitute.
2, timbering material can be degraded in vivo gradually, finally substitute by autologous osseous tissue, catabolite can utilize by self osteogenetic process, and degradation speed and self osteogenetic process can keep synchronous.
3, recombinant human bone morphorgenetic protein-2 mature peptide first with slow releasing agent phase compound, after implanting, BMP-2 mature peptide can be slowly released, thus not easily run off, at new bone formation local long term, can promote that the new bone in defect area local is formed fast.
4, biological degradable coral hydroxyapatite artificial bone provided by the present invention is compound freeze-drying prods, can preserve more than 3 years at-20 DEG C, and can preserve more than 2 years at 4 DEG C, room temperature preservation more than 1.5 years, universality is wide, convenient operation and transport.
5, the production cost of biological degradable coral hydroxyapatite artificial bone provided by the present invention is lower, is a kind of safety, efficient, the cheap abundance of source material, is convenient to the bone graft substitute prepared and preserve.
Accompanying drawing explanation
Fig. 1 is the stereoscan photograph of natural coral;
Fig. 2 is the stereoscan photograph of surface through the Corallium Japonicum Kishinouye of hydroxy-apatite petrochemical industry; Amplification is 50;
Fig. 3 is the stereoscan photograph that surface recombination has the Corallium Japonicum Kishinouye of Human Bone Morphogenetic Proteins-4's crystallization;
Fig. 4 is that artificial bone bone provided by the invention is implanted into section picture, shows cortical bone district skeletonization situation;
Fig. 5 is that artificial bone bone provided by the invention is implanted into section picture, shows bone marrow district skeletonization situation;
Fig. 6 is the stereoscan photograph of surface through the Corallium Japonicum Kishinouye of hydroxy-apatite petrochemical industry of high power.Amplification is 3000.
Detailed description of the invention
In order to be more readily understood functional character and the advantage of artificial bone provided by the invention, hereafter by preferred embodiment of the present invention, and coordinate graphic being described below in detail:
The raw material of the support of artificial bone provided by the invention adopts natural coral, as shown in Figure 1.
Surface warp and the (NH of the rack body obtained by natural coral
4)
2hPO
4reaction generates hydroxyapatite degraded layer, obtains the support of artificial bone, as shown in Figure 2.
Shown in Fig. 2, the rack surface compound Human Bone Morphogenetic Proteins-4 of artificial bone and slow releasing agent, as shown in Figure 3, obtain artificial bone of the present invention.Human Bone Morphogenetic Proteins-4 in Fig. 3 is crystal habit.
Can see from Fig. 1, Fig. 2 and Fig. 6, generate hydroxyapatite by the calcium carbonate of natural coral, the coral hydroxyapatite generated after hydro-thermal reaction remains porous and the high porosity of natural coral, and surface texture there occurs obvious change.As shown in Figure 3, described recombinant human bone morphorgenetic protein-2 mature peptide is at the surface of coral hydroxyapatite and the visible protein crystal of hole.
As shown in Figure 4, artificial bone provided by the invention implants White Rabbit cortical bone district, and bone is implanted into local organization reaction no significant difference compared with control material.Control material refers to: hydroxyl apatite bioceramic (state's food medicine prison No. 3461652nd, tool (standard) word 2011).
As shown in Figure 5, artificial bone provided by the invention implants rat bone defective region, and bone is implanted into local organization reaction no significant difference compared with control material.Control material refers to: hydroxyl apatite bioceramic (state's food medicine prison No. 3461652nd, tool (standard) word 2011).
3m in Fig. 4 and Fig. 5 refers to 3 months, and 6m refers to 6 months.
In Fig. 4, the picture that 3m is corresponding is that artificial bone bone is implanted into section picture, shows the cortical bone district skeletonization situation that artificial bone is implanted 3 months.The picture that 6m is corresponding is that artificial bone bone is implanted into section picture, shows the cortical bone district skeletonization situation that artificial bone is implanted 6 months.
In Fig. 5, the picture that 3m is corresponding is that artificial bone bone is implanted into section picture, shows the bone marrow district skeletonization situation that artificial bone is implanted 3 months.The picture that 6m is corresponding is that artificial bone bone is implanted into section picture, shows the bone marrow district skeletonization situation that artificial bone is implanted 6 months.
Biological degradable coral hydroxyapatite artificial bone is prepared according to following method provided by the invention.The method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; Cleaning; Baking oven is put at 50-100 DEG C of dry 3-4 hour after cleaning up; Then, by Corallium Japonicum Kishinouye and (NH
4)
2hPO
4aqueous solution together put into autoclave, (NH
4)
2hPO
4the mass concentration of aqueous solution is 0.06mol/L, (NH
4)
2hPO
4(NH in solution
4)
2hPO
4with Corallium Japonicum Kishinouye (CaCO
3) mol ratio be 1:10; Again to (NH
4)
2hPO
4potassium dihydrogen phosphate is added, often liter of (NH in solution
4)
2hPO
4potassium dihydrogen phosphate 1mg is added in solution; Pressure in reactor is 7MP, and temperature is 280 DEG C; React 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Drying is distributed into cillin bottle, finally uses gamma-rays illumination-based disinfection again.
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the aqueous solution of concentration 0.1-1mg/ml, the slow releasing agent accounting for aqueous solution gross weight 0.2%-2% is added in aqueous solution, under low temperature, low speed is stirred to mixing fully, obtains the homogeneous mixture solotion of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite support
Be added to by the mixed solution that step (2) obtains in degradable coral hydroxyapatite support that step (1) obtains, addition is 0.1-1mg/cm
3(that is: every cubic centimetre of support adds the mixed solution containing 0.1-1mg recombinant human bone morphorgenetic protein-2 mature peptide), low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent fully enter in the hole of degradable coral hydroxyapatite support, less than-30 DEG C evacuation, lyophilization, obtain described artificial bone, artificial bone is sealed, preserves for a long time for-20 DEG C.
In step (1), the main component of natural coral is calcium carbonate (CaCO
3), warp and (NH
4)
2hPO
4reaction can make surface portion be converted into hydroxyapatite.
Embodiment 1
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is the combination of collagen and fibronectin, and the mass ratio of collagen and fibronectin is 1:1.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV QTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR (see sequence 1).
Described artificial bone is grain type, and diameter is 2-6mm, and the pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
The concentration of described bone-inducing activity cytokine is 0.6%mg/cm
3, the concentration of described slow releasing agent is 0.2%.
By the artificial bone that embodiment 1 prepares, censorship is surveyed unit and is detected.Detection unit is: Nat'l Pharmaceutical & Biological Products Control Institute.Survey report is numbered: QZ200701428.Inspection item: partial test.Main Detection of content and result are see table 1 below.
The testing result of the artificial bone that table 1 embodiment 1 prepares
Embodiment 2
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is selected from collagen and hyaluronic combination, and collagen and hyaluronic mass ratio are 1:1.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。(see sequence 1)
Described support is grain type, and diameter is 5-6mm, and the pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.The concentration of described bone-inducing activity cytokine is 1mg/cm
3, the concentration of described slow releasing agent is 0.2%.
By the artificial bone that embodiment 2 prepares, censorship is surveyed unit and is detected.Detection unit is: Nat'l Pharmaceutical & Biological Products Control Institute.Survey report is numbered: QZ200804480.Inspection item: Implantation Test.Main Detection of content and result are see table 2 below.
The Implantation Test result of the artificial bone that embodiment 2 prepares by table 2
Nat'l Pharmaceutical & Biological Products Control Institute's artificial bone is implanted into section, as shown in Figure 4 and Figure 5.
Embodiment 3
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is the combination of hyaluronic acid and polyvinylpyrrolidone, and the mass ratio of hyaluronic acid and polyvinylpyrrolidone is 1:1.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。(see sequence 1)
Described artificial bone is grain type, and diameter is 4-6mm, and the pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
The concentration of described bone-inducing activity cytokine is 0.8mg/cm
3, the concentration of described slow releasing agent is 0.2%.
Embodiment 4
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is collagen.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。
Described artificial bone is column type: body diameter is 10 ± 0.5, and cylinder is high is 20 ± 1.0.The pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
The concentration of described bone-inducing activity cytokine is 0.6mg/cm
3, the concentration of described slow releasing agent is 1%.
Embodiment 5
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is hyaluronic acid.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。(see sequence 1)
Described artificial bone is column type: body diameter is 10 ± 0.5, and cylinder is high is 20 ± 1.0.The pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
The concentration of described bone-inducing activity cytokine is 1mg/cm
3, the concentration of described slow releasing agent is 2%.
Embodiment 6
The invention provides a kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is collagen, fibronectin, hyaluronic acid and chitinous combination, and collagen, fibronectin, hyaluronic acid and chitinous mass ratio are 1:1:1:1.
The aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。(see sequence 1)
Described artificial bone is column type: body diameter is 10 ± 0.5, and cylinder is high is 20 ± 1.0.The pore diameter range of described micropore is 77-180 μm, and the porosity of described micropore is 8%-11%.
The concentration of described bone-inducing activity cytokine is 0.8mg/cm
3, the concentration of described slow releasing agent is 1.5%.
The artificial bone prepared by embodiment 3-6, censorship is surveyed unit and is detected.Detection unit is: National Institute for Food and Drugs Control.Survey report is numbered: QZ201206312.Inspection item: partial test.Main Detection of content and result are see table 3 below.
The testing result of the artificial bone that table 3 embodiment 3-6 prepares
Above-mentioned examining report with reference to following regulatory documents:
YY 0305-1998 hydroxyl apatite bioceramic
GB/T 16886-1997 BiologicalEvaluationofMedicalDevice
The Pharmacopoeia of the People's Republic of China three general rules and annex: biological product chemistry and other calibration methods.
Comparative example 1
A kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises timbering material, described timbering material is attached with bone-inducing activity cytokine, slow releasing agent; Described timbering material is degradable coral hydroxyapatite; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of described bone-inducing activity cytokine is 2mg/cm
3; Described slow releasing agent is collagen, and the concentration of slow releasing agent is 0.3%.
The preparation method of above-mentioned degradable coral hydroxyapatite artificial bone, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; First ultrasonic cleaning, rear flowing water cleans 20 minutes, to remove organic principle and Corallium Japonicum Kishinouye chip in Surface of Coral and internal void; Baking oven is put at 60 DEG C dry 4 hours after cleaning up; Then, 100 grams of Corallium Japonicum Kishinouyes handled well and 8.1 grams of (NH are got
4)
2hPO
4putting into the autoclave of 2L together with 1L deionized water, is 280 DEG C in temperature, and pressure is under 7MPa, reacts 12 hours, and take out product, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Drying is distributed into cillin bottle, finally uses gamma-rays illumination-based disinfection again;
(2) complex liquid of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent is prepared
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the aqueous solution of concentration 2mg/ml, and add wherein and account for aqueous solution gross weight 0.3% slow releasing agent, under low temperature, low speed is stirred to and dissolves completely, and cryogenic temperature is 4 DEG C, and stirring at low speed rotating speed is 100 turns/min; After solution left standstill to room temperature, with 0.22 μm of membrane filtration, disinfecting container is collected, and becomes the composite solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite
In the degradable coral hydroxyapatite material that step (1) obtains, according to 2mg/cm
3add the solution that step (2) obtains, and put it in Sterile vacuum exsiccator, aspirate 10 minutes at 4 DEG C, the complex liquid of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent is made to be inhaled in degradable coral hydroxyapatite hole completely, the cillin bottle this being filled degradable coral hydroxyapatite and mature peptide of human bone morphogenetic protein-2 complex moves in freeze dryer,-35 DEG C of evacuation lyophilizations, sealing, product-20 DEG C preservation.
Comparative example 2
A kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises timbering material, described timbering material is attached with bone-inducing activity cytokine, slow releasing agent; Described timbering material is degradable coral hydroxyapatite; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of described bone-inducing activity cytokine is 3mg/cm
3; Described slow releasing agent is collagen, and the concentration of slow releasing agent is 0.4%.
Comparative example 3
A kind of biological degradable coral hydroxyapatite artificial bone, this artificial bone comprises timbering material, described timbering material is attached with bone-inducing activity cytokine, slow releasing agent; Described timbering material is degradable coral hydroxyapatite; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of described bone-inducing activity cytokine is 0.5mg/cm
3; Described slow releasing agent is collagen, and the concentration of slow releasing agent is 0.1%.
Comparative example 4
A preparation method for degradable coral hydroxyapatite artificial bone, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; First ultrasonic cleaning, rear flowing water cleans; Baking oven is put at 50 DEG C dry 4 hours after cleaning up; Then, 95 grams of Corallium Japonicum Kishinouyes handled well and 5 grams of (NH are got
4)
2hPO
4putting into the autoclave of 2L together with 1L deionized water, is 270 DEG C in temperature, and pressure is under 9MPa, and react 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Drying is distributed into cillin bottle, finally uses gamma-rays illumination-based disinfection again;
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the aqueous solution of concentration 1mg/ml, adds 0.1% slow releasing agent wherein, and under low temperature, low speed is stirred to and dissolves completely, and cryogenic temperature is 2 DEG C, and stirring at low speed rotating speed is 150 turns/min; After solution left standstill to room temperature, with 0.22 μm of membrane filtration, disinfecting container is collected, and becomes the homogeneous solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite
In the degradable coral hydroxyapatite material that step (1) obtains, according to 1mg/cm
3add the solution that step (2) obtains, at 1 DEG C, negative pressure is under-0.01MPa, the mixed liquor of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent is made fully to enter in degradable coral hydroxyapatite hole, less than-30 DEG C evacuation lyophilizations, sealing, preserves for-20 DEG C for a long time.
Comparative example 5
The preparation method of this kind of degradable coral hydroxyapatite artificial bone, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; First ultrasonic cleaning, rear flowing water cleans; Baking oven is put at 100 DEG C dry 3 hours after cleaning up; Then, 75 grams of Corallium Japonicum Kishinouyes handled well and 25 grams of (NH are got
4)
2hPO
4putting into the autoclave of 2L together with 1L deionized water, is 280 DEG C in temperature, and pressure is under 4MPa, and react 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Drying is distributed into cillin bottle, finally uses gamma-rays illumination-based disinfection again;
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the solution of concentration 5mg/ml, adds 0.5% slow releasing agent wherein, and under low temperature, low speed is stirred to and dissolves completely, and cryogenic temperature is 6 DEG C, and stirring at low speed rotating speed is 50 turns/min; After solution left standstill to room temperature, with 0.22 μm of membrane filtration, disinfecting container is collected, and becomes the homogeneous solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite
In the degradable coral hydroxyapatite material that step (1) obtains, according to 5mg/cm
3add the solution that step (2) obtains, at 5 DEG C, negative pressure is under-0.05MPa, the mixed liquor of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent is made fully to enter in degradable coral hydroxyapatite hole, less than-30 DEG C evacuation lyophilizations, sealing, preserves for-20 DEG C for a long time.
The partial properties index of the cmposite artificial bone prepared by embodiment 1-6 and comparative example 1-5 detects, and detects knot and looks into see table 4.
The testing performance index result of table 4 embodiment and the artificial bone prepared by comparative example
Analysis of application result
Get Adult New Zealand White Rabbit 33, be divided into embodiment group (18), comparative example group (15), often kind of artificial bone is used for 3 White Rabbits.Artificially manufacture the round defect district of a diameter 1.5 centimetres on the skull top of White Rabbit, the implantation of embodiment group obtains according to embodiment, and the diameter being polished into chip shape is about the cmposite artificial bone of 1.5cm; Comparative example group is implanted according to obtained by comparative example, and the diameter being polished into chip shape is about the cmposite artificial bone of 1.5cm; Postoperatively carry out periosteum and skin closure, again perform the operation after 10 weeks, observe healing state, comparative example group defect of skull healing area is less than 50%; Embodiment group defect of skull healing area is greater than 50%.Again perform the operation after 3 months, observe healing state, the healing of comparative example group defect of skull is good; Comparatively comparative example group is better for embodiment group defect of skull healing state.Again perform the operation after 28 weeks, observing healing state, heals completely in embodiment group defect of skull position, and comparative example group defect of skull position also has the part that do not heal of about 5-10%.
Take out the freshman bone tissue of 26 weeks of a White Rabbit of embodiment and comparative example respectively, first by scanning electron microscopic observation new bone formation and the fusion situation with surrounding bone tissue, detect the degraded situation of artificial bone scaffold afterwards.
Take off freshman bone tissue, X-ray is observed visible: have a large amount of new bone formation in the defect of skull region of artificial bone reparation provided by the invention, area of new bone and surrounding bone tissue fusion are good, in artificial bone fill area, timbering material major part all by absorption by freshman bone tissue substitute.And area of new bone quantity, area of new bone and the surrounding bone tissue fusion state difference all more of the present invention in the part lack of skull that the artificial bone that comparative example provides is repaired.
Can be drawn by the test result of above-described embodiment and comparative example and applied analysis, biological degradable coral hydroxyapatite artificial bone provided by the invention possesses the active and bone-inducting active of good bone conduction simultaneously.
The above, be only preferred embodiment of the present invention, be not intended to limit protection scope of the present invention.Every equalization done according to content of the present invention changes and modifies, and is all encompassed in the scope of the claims of the present invention.
Claims (10)
1. a biological degradable coral hydroxyapatite artificial bone, is characterized in that, this artificial bone comprises support, described support is attached with bone-inducing activity cytokine, slow releasing agent; Described support comprises rack body, and the body of support is Corallium Japonicum Kishinouye, and rack body surface coverage has hydroxyapatite layer; Described bone-inducing activity cytokine is restructuring mature peptide of human bone morphogenetic protein-2; Described slow releasing agent is selected from the combination of a kind of in collagen, fibronectin, hyaluronic acid, chitin, polyvinylpyrrolidone or at least two kinds.
2. degradable coral hydroxyapatite artificial bone according to claim 1, it is characterized in that, described support is irregular 3-D solid structure, and the scope of length, width and height is 2-20mm, the pore diameter range of described micropore is 77-200 μm, and the porosity of described micropore is 8%-11%.
3. degradable coral hydroxyapatite artificial bone according to claim 1, it is characterized in that, the aminoacid sequence of described recombinant human bone morphorgenetic protein-2 mature peptide is as follows:
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR。
4. degradable coral hydroxyapatite artificial bone according to claim 1, it is characterized in that, the concentration of described bone-inducing activity cytokine is 0.1-1mg/cm
3, the concentration of described slow releasing agent is 0.2-2%.
5. degradable coral hydroxyapatite artificial bone according to claim 1, it is characterized in that, the concentration of described bone-inducing activity cytokine is 0.6-1mg/cm
3, the concentration of described slow releasing agent is 0.2%.
6. degradable coral hydroxyapatite artificial bone according to claim 1, it is characterized in that, the concentration of described bone-inducing activity cytokine is 0.6-1mg/cm
3, the concentration of described slow releasing agent is 1-2%.
7. according to the preparation method of the described degradable coral hydroxyapatite artificial bone of one of claim 1 to 6, it is characterized in that, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; Cleaning; Baking oven is put at 50-100 DEG C of dry 3-4 hour after cleaning up; Then, by Corallium Japonicum Kishinouye and (NH
4)
2hPO
4aqueous solution together put into autoclave, (NH
4)
2hPO
4the mass concentration of aqueous solution is 0.05-1mol/L, (NH
4)
2hPO
4(NH in solution
4)
2hPO
4with Corallium Japonicum Kishinouye (CaCO
3) mol ratio be 1:10-15; Again to (NH
4)
2hPO
4potassium dihydrogen phosphate is added, often liter of (NH in solution
4)
2hPO
4potassium dihydrogen phosphate 1mg is added in solution; React 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Use gamma-rays illumination-based disinfection again, obtain degradable coral hydroxyapatite support;
(2) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are mixed with the aqueous solution of mixing homogeneously;
(3) in surface recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent being attached to degradable coral hydroxyapatite support and hole.
8. according to the preparation method of the described degradable coral hydroxyapatite artificial bone of one of claim 1-6, it is characterized in that, described method comprises the steps:
(1) degradable coral hydroxyapatite is prepared by Corallium Japonicum Kishinouye
Get Corallium Japonicum Kishinouye and carry out cutting polishing, be prepared into required size, shape; Cleaning; Baking oven is put at 50-100 DEG C of dry 3-4 hour after cleaning up; Then, by Corallium Japonicum Kishinouye and (NH
4)
2hPO
4aqueous solution together put into autoclave, (NH
4)
2hPO
4the mass concentration of aqueous solution is 0.05-1mol/L, (NH
4)
2hPO
4(NH in solution
4)
2hPO
4with Corallium Japonicum Kishinouye (CaCO
3) mol ratio be 1:10-1:15; Again to (NH
4)
2hPO
4potassium dihydrogen phosphate is added, often liter of (NH in solution
4)
2hPO
4potassium dihydrogen phosphate 1mg is added in solution; React 12 hours, boil 4 hours with deionized water afterwards, per half an hour changes deionized water once, removes inorganic ions; Use gamma-rays illumination-based disinfection again, obtain degradable coral hydroxyapatite support, finally by dry for degradable coral hydroxyapatite support subpackage cillin bottle;
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent
Recombinant human bone morphorgenetic protein-2 mature peptide is mixed with the aqueous solution of concentration 0.1-1mg/ml, the slow releasing agent accounting for aqueous solution gross weight 0.2%-2% is added in aqueous solution, under low temperature, low speed is stirred to mixing fully, obtains the homogeneous mixture solotion of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent;
(3) recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent are attached on degradable coral hydroxyapatite support
Be added to by the mixed solution that step (2) obtains in degradable coral hydroxyapatite support that step (1) obtains, addition is 0.1-1mg/cm
3(that is: every cubic centimetre of support adds the mixed solution containing 0.1-1mg recombinant human bone morphorgenetic protein-2 mature peptide), low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and slow releasing agent fully enter in the hole of degradable coral hydroxyapatite support, less than-30 DEG C evacuation, lyophilization, obtain described artificial bone, artificial bone is sealed, preserves for a long time for-20 DEG C.
9. the preparation method of degradable coral hydroxyapatite artificial bone according to claim 7 or 8, is characterized in that, the pressure in described step (1) in reactor is: 4-9MPa, temperature is: 270-280 DEG C.
10. the preparation method of degradable coral hydroxyapatite artificial bone according to claim 7 or 8, is characterized in that, Corallium Japonicum Kishinouye and (NH in described step (1)
4)
2hPO
4ratio be: 10:1, the pressure in reactor is: 7MP, and temperature is: 280 DEG C.
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| CN111388754A (en) * | 2020-04-09 | 2020-07-10 | 珠海深泓鑫生物科技有限公司 | Bioactive artificial bone material and preparation method thereof |
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| CN106806940A (en) * | 2015-12-02 | 2017-06-09 | 中国科学院大连化学物理研究所 | A kind of preparation method of nano hydroxylapatite doped porous Bionics Bone support |
| CN111388754A (en) * | 2020-04-09 | 2020-07-10 | 珠海深泓鑫生物科技有限公司 | Bioactive artificial bone material and preparation method thereof |
| CN113952504A (en) * | 2021-10-29 | 2022-01-21 | 华南理工大学 | Selenium-doped coral/hydroxyapatite scaffold and preparation method thereof |
| CN113952504B (en) * | 2021-10-29 | 2022-12-16 | 华南理工大学 | Selenium-doped coral/hydroxyapatite scaffold and preparation method thereof |
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