CN104474591B - Bioactive degradable coral hydroxyapatite artificial bone - Google Patents
Bioactive degradable coral hydroxyapatite artificial bone Download PDFInfo
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- CN104474591B CN104474591B CN201410843797.1A CN201410843797A CN104474591B CN 104474591 B CN104474591 B CN 104474591B CN 201410843797 A CN201410843797 A CN 201410843797A CN 104474591 B CN104474591 B CN 104474591B
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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, more particularly to a kind of biological degradable coral hydroxy phosphorus ash
Stone 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 cannot pass through human body self-recovery machine
Repair deficiency processed, it is therefore necessary to carry out bone collection treatment.
Material for bone collection treatment is referred to as artificial bone, and artificial bone needs suitably sized, suitable porosity
And aperture, and with suitable material and function.
Preferably bone grafting material, needs to be provided simultaneously with osteoacusis and bone-inducting active.Coral and by derived from coral hydroxyl
Base Apatite materials have and pore structure as human body bone photo, and as timbering material the filling of Cranial defect is widely used in, because
It has good 3-D solid structure and biomechanical property, therefore shows outstanding osteoacusis activity, but area of new bone
Inducibility is not enough, and osteogenetic process is slow, and particularly with big intercept Cranial defect and large area Cranial defect satisfied treatment cannot be obtained
Effect.In addition, coral main component CaCO3, implant and be degraded quickly, it is not enough to maintain whole osteogenetic process, and hydroxyl phosphorus
Lime stone degradation in vivo speed is extremely slow, affects the replacement of autologous tissue.BMP-2 is that have most to induce bone by force in human body
One of cell factor of Forming ability, the use of BMP-2 can with accelerating union of bone fracture and new bone formation process, but,
BMP-2 is small protein, easily diffusion, it is impossible to enough that valid density is kept in the region for need skeletonization, is continued
Play bone inductive effect, although therefore cell factor with good bone-inducing activity, exclusive use still cannot repairing in Cranial defect
Positive effect is played during multiple.
Therefore, for tissue engineering artificial bone, the use of single key element is difficult to meet and clinically bone collection is replaced
For the demand of thing, at present research and using the compound use for progressively having turned to many key elements, to build more effective more reasonably people
Work bone.
The content of the invention
In order to the degradability for solving existing artificial bone is undesirable, the poor defect of bone-inducting active, the present invention provides one
Plant biological degradable coral hydroxyapatite artificial bone and preparation method thereof.
In order to solve above-mentioned technical problem, the present invention adopts following technical proposals：
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；One kind in collagen, fibronectin, hyaluronic acid, chitin, the polyvinylpyrrolidone of described sustained release agent or at least
Two kinds of combination.
Further, the support is irregular 3-D solid structure, and the scope of length, width and height is 2-
20mm, the pore diameter range of the micropore is 77-200 μm, and the porosity of the micropore is 8%-11%.
Further, the artificial bone is grain type or column type.
Further, grain type artificial bone：A diameter of 2-6mm.
Further, column type artificial bone：Body diameter is 10 ± 0.5mm；A height of 20 ± the 1.0mm of cylinder.
Further, the pore diameter range of the micropore is 77-180 μm, and the porosity of the micropore is 8%-11%.
Further, the amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR.(referring to sequence 1)
Further, the concentration of the bone-inducing activity cell factor is 0.1-1mg/cm3, the concentration of the sustained release agent is
0.2-2%.
The concentration of the bone-inducing activity cell factor is 0.1-1mg/cm3, refer in the support of artificial bone per cubic centimeter
Containing 0.1-1mg bone-inducing activity cell factors.The concentration of the sustained release agent is 0.2-2%, is referred to per 100 cubic centimetres of artificial bones
Support be contained within 0.2-2g sustained release agents.
Further, the concentration of the bone-inducing activity cell factor is 0.6-1mg/cm3, the concentration of the sustained release agent is
0.2%.
Further, the concentration of the bone-inducing activity cell factor is 0.6-1mg/cm3, the concentration of the sustained release agent is 1-
2%.
Further, described sustained release agent be collagen, fibronectin, hyaluronic acid and chitinous combination, aforementioned four kinds
The mass ratio of sustained release agent is 1:1:1:1.
The present invention also provides the preparation method of above-mentioned degradable coral hydroxyapatite artificial bone, and methods described includes following
Step：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；Cleaning；Baking oven is put into after cleaning up in 50-
100 DEG C are dried 3-4 hours；Then, by coral and (NH4)2HPO4The aqueous solution be together put into autoclave, (NH4)2HPO4Water
The mass concentration of solution is 0.05-1mol/L, (NH4)2HPO4(NH in solution4)2HPO4With coral (CaCO3) mol ratio be
1:10-15；Again to (NH4)2HPO4Potassium dihydrogen phosphate, per liter of (NH are added in solution4)2HPO4Potassium dihydrogen phosphate is added in solution
1mg；Reaction 12 hours, afterwards deionized water boil 4 hours, change deionized water once per half an hour, remove inorganic ions；Again
Gamma-rays illumination-based disinfection is used, degradable coral hydroxyapatite support is obtained；
(2) recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent are configured to the aqueous solution being well mixed.
(3) recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent are attached to into degradable coral hydroxyapatite support
In surface and hole.
Further, the preparation method of the degradable coral hydroxyapatite artificial bone, methods described includes following steps
Suddenly：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；Cleaning；Baking oven is put into after cleaning up in 50-
100 DEG C are dried 3-4 hours；Then, by coral and (NH4)2HPO4The aqueous solution be together put into autoclave, (NH4)2HPO4Water
The mass concentration of solution is 0.05-1mol/L, (NH4)2HPO4(NH in solution4)2HPO4With coral (CaCO3) mol ratio be
1:10-1:15；Again to (NH4)2HPO4Potassium dihydrogen phosphate, per liter of (NH are added in solution4)2HPO4Potassium dihydrogen phosphate is added in solution
1mg；Reaction 12 hours, afterwards deionized water boil 4 hours, change deionized water once per half an hour, remove inorganic ions；Again
Gamma-rays illumination-based disinfection is used, degradable coral hydroxyapatite support is obtained, finally by degradable coral hydroxyapatite support
It is dried packing cillin bottle；
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the aqueous solution of concentration 0.1-1mg/ml, adds in aqueous solution
Enter to account for the sustained release agent of aqueous solution gross weight 0.2%-2%, stirring at low speed is abundant to mixing under low temperature, obtain recombinant human bone and form egg
The homogeneous mixture solotion of -2 mature peptides and sustained release agent in vain；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite support with sustained release agent
The mixed solution that step (2) is obtained is added in the degradable coral hydroxyapatite support that step (1) is obtained, plus
Enter amount for 0.1-1mg/cm3(i.e.：Support per cubic centimeter is added containing 0.1-1mg recombinant human bone morphorgenetic protein-2 mature peptides
Mixed solution), low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent well into degradable
In the hole of coral hydroxyapatite support, less than -30 DEG C vacuumize, freeze-drying, described artificial bone are obtained, by artificial bone
Sealing, -20 DEG C long-term preserve.
The main component of natural coral is calcium carbonate (CaCO in step (1)3), Jing and (NH4)2HPO4Reaction can make surface
It is partially converted into hydroxyapatite.
Further, the cleaning process in step (1) be first be cleaned by ultrasonic, after with flowing water rinse 20 minutes, with remove
Organic principle and coral debris in Surface of Coral and internal void.
Further, in the preparation method of the degradable coral hydroxyapatite artificial bone, reaction in the step (1)
Pressure in kettle is：4-9MPa, temperature is：270-280℃.
Further, in the preparation method of the degradable coral hydroxyapatite artificial bone, coral in the step (1)
With (NH4)2HPO4Ratio be：10:1, the pressure in reactor is：7MP, temperature is：280℃.
Further, cryogenic temperature is 2 DEG C -6 DEG C in the step (2), and stirring at low speed rotating speed is 50-150 rev/min.
Further, cryogenic temperature is 1-5 DEG C in described step (3), and negative pressure is -0.01 to -0.05MPa.
Compared with prior art, under biological degradable coral hydroxyapatite artificial bone provided by the present invention has
State advantage：
1st, be provided simultaneously with good osteoacusis activity and bone-inducting active, i.e., with good biomechanics characteristic and from
Pore structure as body bone photo and good histocompatbility, can give full play to the induction area of new bone shape of BMP-2 again
Into ability.It is a kind of preferable bone graft substitute with good bone defect healing ability.
2nd, timbering material can gradually degrade in vivo, finally be substituted by autologous bone tissue, and catabolite can be by itself
Osteogenetic process is utilized, and degradation speed can keep synchronous with itself osteogenetic process.
3rd, recombinant human bone morphorgenetic protein-2 mature peptide is mutually combined first with sustained release agent, the BMP-2 after implanting
Mature peptide can be slowly released, and so as to not easily run off, can promote defect area local in new bone formation local long term
New bone is quickly formed.
4th, biological degradable coral hydroxyapatite artificial bone provided by the present invention, is to be combined freeze-drying prods, energy
It is enough to preserve more than 3 years at -20 DEG C, can preserve more than 2 years at 4 DEG C, room temperature preservation more than 1.5 years, universality is wide, is easy to operation
And transport.
5th, the production cost of biological degradable coral hydroxyapatite artificial bone provided by the present invention is relatively low, is one
Plant safe efficient, source material inexpensively sufficient, be easy to the bone graft substitute for preparing and preserving.
Description of the drawings
Fig. 1 is the stereoscan photograph of natural coral；
Fig. 2 is the stereoscan photograph of the coral of surface Jing hydroxy-apatite petrochemical industry；Multiplication factor is 50；
Fig. 3 has the stereoscan photograph of the coral of Human Bone Morphogenetic Proteins-4's crystallization for surface recombination；
Fig. 4 is implanted into section picture for the artificial bone bone that the present invention is provided, it is shown that cortex bone area skeletonization situation；
Fig. 5 is implanted into section picture for the artificial bone bone that the present invention is provided, it is shown that marrow area skeletonization situation；
Fig. 6 is the stereoscan photograph of the coral of the surface Jing hydroxy-apatite petrochemical industry of high power.Multiplication factor is 3000.
Specific embodiment
In order to be more readily understood the functional character and advantage of the artificial bone of present invention offer, hereafter by the preferably real of the present invention
Example is applied, and coordinates schema to be described below in detail：
The raw material of the support of the artificial bone that the present invention is provided adopts natural coral, as shown in Figure 1.
The surface Jing and (NH of rack body by obtained in natural coral4)2HPO4Reaction generates hydroxyapatite degraded layer,
The support of artificial bone is obtained, as shown in Figure 2.
Human Bone Morphogenetic Proteins-4 and sustained release agent are combined in the rack surface of artificial bone shown in Fig. 2, as shown in figure 3, obtaining this
Bright described artificial bone.Human Bone Morphogenetic Proteins-4 in Fig. 3 is crystal habit.
Can see from Fig. 1, Fig. 2 and Fig. 6, hydroxyapatite be generated by the calcium carbonate of natural coral, through hydro-thermal
The coral hydroxyapatite generated after reaction remains the porous and high porosity of natural coral, and surface texture there occurs substantially
Change.As shown in figure 3, the recombinant human bone morphorgenetic protein-2 mature peptide is visible on the surface of coral hydroxyapatite and hole
Protein crystal.
As shown in figure 4, the artificial bone implantation White Rabbit cortex bone area that the present invention is provided, bone be implanted into local organization reaction with
Control material compares no significant difference.Control material is referred to：Hydroxyl apatite bioceramic (state food medicine prison tool (standard) word 2,011 the
No. 3461652).
As shown in figure 5, the artificial bone implantation rat bone defective region that the present invention is provided, bone be implanted into local organization reaction with it is right
No significant difference is compared according to material.Control material is referred to：Hydroxyl apatite bioceramic (state food medicine prison tool (standard) word 2,011 the
No. 3461652).
3m in Fig. 4 and Fig. 5 refers to that 6m refers to 6 months 3 months.
In Fig. 4, the corresponding pictures of 3m are that artificial bone bone is implanted into section picture, it is shown that artificial bone is implanted into the skin of 3 months
Zhi Gu areas skeletonization situation.The corresponding pictures of 6m are that artificial bone bone is implanted into section picture, it is shown that artificial bone is implanted into the skin of 6 months
Zhi Gu areas skeletonization situation.
In Fig. 5, the corresponding pictures of 3m are that artificial bone bone is implanted into section picture, it is shown that artificial bone is implanted into the bone of 3 months
Marrow area skeletonization situation.The corresponding pictures of 6m are that artificial bone bone is implanted into section picture, it is shown that artificial bone is implanted into the marrow of 6 months
Area's skeletonization situation.
The following methods provided according to the present invention prepare biological degradable coral hydroxyapatite artificial bone.The method
Comprise the steps：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；Cleaning；Baking oven is put into after cleaning up in 50-
100 DEG C are dried 3-4 hours；Then, by coral and (NH4)2HPO4The aqueous solution be together put into autoclave, (NH4)2HPO4Water
The mass concentration of solution is 0.06mol/L, (NH4)2HPO4(NH in solution4)2HPO4With coral (CaCO3) mol ratio be 1:
10；Again to (NH4)2HPO4Potassium dihydrogen phosphate, per liter of (NH are added in solution4)2HPO4Potassium dihydrogen phosphate 1mg is added in solution；Instead
The pressure in kettle is answered to be 7MP, temperature is 280 DEG C；Reaction 12 hours, afterwards deionized water boil 4 hours, change per half an hour
Ionized water once, removes inorganic ions；Drying is distributed into cillin bottle, and gamma-rays illumination-based disinfection is finally used again.
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the aqueous solution of concentration 0.1-1mg/ml, adds in aqueous solution
Enter to account for the sustained release agent of aqueous solution gross weight 0.2%-2%, stirring at low speed is abundant to mixing under low temperature, obtain recombinant human bone and form egg
The homogeneous mixture solotion of -2 mature peptides and sustained release agent in vain；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite support with sustained release agent
The mixed solution that step (2) is obtained is added in the degradable coral hydroxyapatite support that step (1) is obtained, plus
Enter amount for 0.1-1mg/cm3(i.e.：Support per cubic centimeter is added containing 0.1-1mg recombinant human bone morphorgenetic protein-2 mature peptides
Mixed solution), low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent well into degradable
In the hole of coral hydroxyapatite support, less than -30 DEG C vacuumize, freeze-drying, described artificial bone are obtained, by artificial bone
Sealing, -20 DEG C long-term preserve.
The main component of natural coral is calcium carbonate (CaCO in step (1)3), Jing and (NH4)2HPO4Reaction can make surface
It is partially converted into hydroxyapatite.
Embodiment 1
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；It is 1 that described sustained release agent is the mass ratio of the combination of collagen and fibronectin, collagen and fibronectin:1.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR (referring to sequence 1).
The artificial bone be grain type, a diameter of 2-6mm, the pore diameter range of the micropore is 77-180 μm, the micropore
Porosity is 8%-11%.
The concentration of the bone-inducing activity cell factor is 0.6%mg/cm3, the concentration of the sustained release agent is 0.2%.
The artificial bone that embodiment 1 is prepared, censorship is surveyed unit and is detected.Detect that unit is：Chinese medicine is biological
Product examines and determine institute.Survey report is numbered：QZ200701428.Inspection project：Partial test.Predominantly detect content and result referring to
Table 1 below.
The testing result of the artificial bone that the embodiment 1 of table 1 is prepared
Embodiment 2
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；It is 1 that described sustained release agent is selected from the mass ratio of the combination of collagen and hyaluronic acid, collagen and hyaluronic acid:1.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR.(referring to sequence 1)
The support be grain type, a diameter of 5-6mm, the pore diameter range of the micropore is 77-180 μm, the hole of the micropore
Gap rate is 8%-11%.The concentration of the bone-inducing activity cell factor is 1mg/cm3, the concentration of the sustained release agent is 0.2%.
The artificial bone that embodiment 2 is prepared, censorship is surveyed unit and is detected.Detect that unit is：Chinese medicine is biological
Product examines and determine institute.Survey report is numbered：QZ200804480.Inspection project：Implantation Test.Predominantly detect content and result referring to
Table 2 below.
The Implantation Test result of the artificial bone that table 2 prepares embodiment 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 present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；Described sustained release agent is the matter of the combination of hyaluronic acid and polyvinylpyrrolidone, hyaluronic acid and polyvinylpyrrolidone
Amount is than being 1:1.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR.(referring to sequence 1)
The artificial bone be grain type, a diameter of 4-6mm, the pore diameter range of the micropore is 77-180 μm, the micropore
Porosity is 8%-11%.
The concentration of the bone-inducing activity cell factor is 0.8mg/cm3, the concentration of the sustained release agent is 0.2%.
Embodiment 4
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；Described sustained release agent is collagen.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR。
The artificial bone is column type：Body diameter is 10 ± 0.5, cylinder a height of 20 ± 1.0.The aperture model of the micropore
It is 77-180 μm to enclose, and the porosity of the micropore is 8%-11%.
The concentration of the bone-inducing activity cell factor is 0.6mg/cm3, the concentration of the sustained release agent is 1%.
Embodiment 5
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；Described sustained release agent is hyaluronic acid.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR.(referring to sequence 1)
The artificial bone is column type：Body diameter is 10 ± 0.5, cylinder a height of 20 ± 1.0.The aperture model of the micropore
It is 77-180 μm to enclose, and the porosity of the micropore is 8%-11%.
The concentration of the bone-inducing activity cell factor is 1mg/cm3, the concentration of the sustained release agent is 2%.
Embodiment 6
The present invention provides a kind of biological degradable coral hydroxyapatite artificial bone, and the artificial bone includes support, institute
State and be attached with support bone-inducing activity cell factor, sustained release agent；The support includes rack body, and the body of support is coral,
Rack body surface is coated with hydroxyapatite layer；The bone-inducing activity cell factor is that recombinant human bone morphorgenetic protein-2 is ripe
Peptide；Described sustained release agent be collagen, fibronectin, hyaluronic acid and chitinous combination, collagen, fibronectin, hyaluronic acid
It is 1 with chitinous mass ratio:1:1:1.
The amino acid sequence of the recombinant human bone morphorgenetic protein-2 mature peptide is as follows：
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTEL
SAISMLYLDENEKVVLKNYQDMVVEGCGCR.(referring to sequence 1)
The artificial bone is column type：Body diameter is 10 ± 0.5, cylinder a height of 20 ± 1.0.The aperture model of the micropore
It is 77-180 μm to enclose, and the porosity of the micropore is 8%-11%.
The concentration of the bone-inducing activity cell factor is 0.8mg/cm3, the concentration of the sustained release agent is 1.5%.
The artificial bone that embodiment 3-6 is prepared, censorship is surveyed unit and is detected.Detect that unit is：Chinese food medicine
Research institute is determined in product examine.Survey report is numbered：QZ201206312.Inspection project：Partial test.Predominantly detect content and result ginseng
See below table 3.
The testing result of the artificial bone that embodiment 3-6 of table 3 is prepared
Above-mentioned examining report with reference to following regulatory documents：
YY 0305-1998 hydroxyl apatite bioceramics
GB/T 16886-1997 BiologicalEvaluationofMedicalDevices
《Pharmacopoeia of People's Republic of China》Three general rules and annex：Biological products chemistry and other calibration methods.
Comparative example 1
A kind of biological degradable coral hydroxyapatite artificial bone, the artificial bone includes timbering material, the support
Bone-inducing activity cell factor, sustained release agent are attached with material；The timbering material is degradable coral hydroxyapatite；It is described to lure
Bone active cell factor is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of the bone-inducing activity cell factor is 2mg/cm3；
Described sustained release agent is collagen, and the concentration of sustained release agent is 0.3%.
The preparation method of above-mentioned degradable coral hydroxyapatite artificial bone, methods described comprises the steps：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；First be cleaned by ultrasonic, after cleaned with flowing water 20 minutes,
Organic principle and coral debris in remove Surface of Coral and internal void；Baking oven is put into after cleaning up 4 are dried at 60 DEG C
Hour；Then, 100 grams of corals handled well and 8.1 grams of (NH are taken4)2HPO4The reaction under high pressure of 2L is put into together with 1L deionized waters
Kettle, is 280 DEG C in temperature, and pressure is under 7MPa, to react 12 hours, takes out product, and afterwards deionized water boils 4 hours, often
Half an hour changes deionized water once, removes inorganic ions；Drying is distributed into cillin bottle, and gamma-rays illumination-based disinfection is finally used again；
(2) complex liquid of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent is prepared
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the aqueous solution of concentration 2mg/ml, is added thereto to account for the aqueous solution total
The sustained release agent of weight 0.3%, to being completely dissolved, cryogenic temperature is 4 DEG C to stirring at low speed under low temperature, stirring at low speed rotating speed is 100 turns/
min；Solution left standstill to room temperature, with 0.22 μm of membrane filtration, collect, and becomes recombinant human bone morphorgenetic protein-2 ripe by disinfecting container
The composite solution of peptide and sustained release agent；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite with sustained release agent
In the degradable coral hydroxyapatite material obtained to step (1), according to 2mg/cm3Step (2) is added to obtain
Solution, and put it in Sterile vacuum drier, aspirate 10 minutes at 4 DEG C, make recombinant human bone morphorgenetic protein-2 mature peptide
It is inhaled into completely in degradable coral hydroxyapatite hole with the complex liquid of sustained release agent, this is filled into degradable coral hydroxy phosphorus
The cillin bottle of lime stone and mature peptide of human bone morphogenetic protein-2 compound is moved in freeze dryer, and -35 DEG C vacuumize freeze-drying, close
Envelope, -20 DEG C of preservations of product.
Comparative example 2
A kind of biological degradable coral hydroxyapatite artificial bone, the artificial bone includes timbering material, the support
Bone-inducing activity cell factor, sustained release agent are attached with material；The timbering material is degradable coral hydroxyapatite；It is described to lure
Bone active cell factor is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of the bone-inducing activity cell factor is 3mg/cm3；
Described sustained release agent is collagen, and the concentration of sustained release agent is 0.4%.
Comparative example 3
A kind of biological degradable coral hydroxyapatite artificial bone, the artificial bone includes timbering material, the support
Bone-inducing activity cell factor, sustained release agent are attached with material；The timbering material is degradable coral hydroxyapatite；It is described to lure
Bone active cell factor is restructuring mature peptide of human bone morphogenetic protein-2, and the concentration of the bone-inducing activity cell factor is 0.5mg/
cm3；Described sustained release agent is collagen, and the concentration of sustained release agent is 0.1%.
Comparative example 4
A kind of preparation method of degradable coral hydroxyapatite artificial bone, methods described comprises the steps：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；First be cleaned by ultrasonic, after cleaned with flowing water；Cleaning is dry
It is put into baking oven after net to be dried 4 hours at 50 DEG C；Then, 95 grams of corals handled well and 5 grams of (NH are taken4)2HPO4With 1L deionizations
Water is put into together the autoclave of 2L, is 270 DEG C in temperature, and pressure is reaction 12 hours under 9MPa, afterwards deionized water
Boil 4 hours, deionized water is changed once per half an hour, remove inorganic ions；Drying is distributed into cillin bottle, and gamma-rays is finally used again
Illumination-based disinfection；
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the aqueous solution of concentration 1mg/ml, is added thereto to 0.1% sustained release
Agent, to being completely dissolved, cryogenic temperature is 2 DEG C to stirring at low speed under low temperature, and stirring at low speed rotating speed is 150 turns/min；Solution left standstill is extremely
After room temperature, with 0.22 μm of membrane filtration, disinfecting container is collected, and becomes recombinant human bone morphorgenetic protein-2 mature peptide equal with sustained release agent
Even solution；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite with sustained release agent
In the degradable coral hydroxyapatite material obtained to step (1), according to 1mg/cm3Step (2) is added to obtain
Solution, at 1 DEG C, negative pressure is under -0.01MPa, to make recombinant human bone morphorgenetic protein-2 mature peptide abundant with the mixed liquor of sustained release agent
Into in degradable coral hydroxyapatite hole, less than -30 DEG C vacuumize freeze-drying, sealing, and -20 DEG C long-term preserve.
Comparative example 5
A kind of this preparation method of degradable coral hydroxyapatite artificial bone, methods described comprises the steps：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；First be cleaned by ultrasonic, after cleaned with flowing water；Cleaning is dry
It is put into baking oven after net to be dried 3 hours at 100 DEG C；Then, 75 grams of corals handled well and 25 grams of (NH are taken4)2HPO4With 1L go from
Sub- water is put into together the autoclave of 2L, is 280 DEG C in temperature, and pressure is under 4MPa, to react 12 hours, and deionization is used afterwards
Water boils 4 hours, and deionized water is changed once per half an hour, removes inorganic ions；Drying is distributed into cillin bottle, is finally penetrated with γ again
Line illumination-based disinfection；
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the solution of concentration 5mg/ml, is added thereto to 0.5% sustained release agent,
To being completely dissolved, cryogenic temperature is 6 DEG C to stirring at low speed under low temperature, and stirring at low speed rotating speed is 50 turns/min；Solution left standstill is to room temperature
Afterwards, with 0.22 μm of membrane filtration, disinfecting container is collected, and becomes recombinant human bone morphorgenetic protein-2 mature peptide uniform molten with sustained release agent
Liquid；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite with sustained release agent
In the degradable coral hydroxyapatite material obtained to step (1), according to 5mg/cm3Step (2) is added to obtain
Solution, at 5 DEG C, negative pressure is under -0.05MPa, to make recombinant human bone morphorgenetic protein-2 mature peptide abundant with the mixed liquor of sustained release agent
Into in degradable coral hydroxyapatite hole, less than -30 DEG C vacuumize freeze-drying, sealing, and -20 DEG C long-term preserve.
The partial properties index of the cmposite artificial bone that embodiment 1-6 and comparative example 1-5 are prepared is detected, detected
Knot is looked into referring to table 4.
The testing performance index result of the artificial bone prepared by the embodiment of table 4 and comparative example
Analysis of application result
Adult New Zealand White Rabbit 33 is taken, is divided into embodiment group (18), comparative example group (15), every kind of artificial bone is used
In 3 White Rabbits.The round defect area of 1.5 centimetres of a diameter is artificially manufactured on the skull top of White Rabbit, embodiment group is planted
Enter the cmposite artificial bone according to the diameter about 1.5cm for obtained in embodiment, being polished into chip shape；Comparative example group is implanted into according to right
Obtained by ratio, the cmposite artificial bone of the diameter about 1.5cm of chip shape is polished into；It is postoperative to carry out periosteum and skin closure,
Performed the operation again after 10 weeks, observed healing state, comparative example group skull defeci healing area is less than 50%；Embodiment group skull
Defect healing area is more than 50%.Performed the operation again after 3 months, observed healing state, the healing of comparative example group skull defeci is good
It is good；Embodiment group skull defeci healing state is more preferable compared with comparative example group.Performed the operation again after 28 weeks, observed healing state, it is real
Apply example group skull defeci position to heal completely, there be the part of not healing of about 5-10% at comparative example group skull defeci position.
Respectively the freshman bone tissue of 26 weeks of a White Rabbit of taking-up embodiment and comparative example, first uses scanning electron microscopic observation
New bone formation and the fusion situation with bone tissue, detect afterwards the degraded situation of artificial bone scaffold.
Freshman bone tissue is removed, X-ray observation is visible：Have in the skull defeci region of the artificial Bone Defect Repari that the present invention is provided
A large amount of new bone formations, area of new bone and bone tissue good knitting, in artificial bone filling region, timbering material major part is all inhaled
Receipts are substituted by freshman bone tissue.And the area of new bone quantity in the part lack of skull of the artificial Bone Defect Repari that comparative example is provided, it is newborn
Bone and bone tissue Fusion Strain difference all more of the invention.
Can be drawn by the test result and applied analysis of above-described embodiment and comparative example, the biologically active that the present invention is provided
Degradable coral hydroxyapatite artificial bone is provided simultaneously with good osteoacusis activity and bone-inducting active.
The above, only presently preferred embodiments of the present invention is not intended to limit protection scope of the present invention.It is every
The impartial change done according to present invention and modification, are encompassed by the scope of the claims of the present invention.
Claims (9)
1. amino acid sequence is
KRLKSSCKRHPLYVDFSDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCV
The recombinant human bone morphorgenetic protein-2 mature peptide of PTELSAISMLYLDENEKVVLKNYQDMVVEGCGCR can in preparation biologically active
Application in degraded coral hydroxyapatite artificial bone, it is characterised in that the artificial bone includes support, is attached with the support
Bone-inducing activity cell factor and sustained release agent；The support includes rack body, and the body of support is coral, and rack body surface is covered
It is stamped hydroxyapatite layer；The bone-inducing activity cell factor is the recombinant human bone morphorgenetic protein-2 mature peptide；Described is slow
Release the one kind or at least two combination of agent in collagen, fibronectin, hyaluronic acid, chitin, polyvinylpyrrolidone.
2. application according to claim 1, it is characterised in that the support be irregular 3-D solid structure, length,
The scope of width and height is 2-20mm, and the pore diameter range of the micropore of support is 77-200 μm, the porosity of the micropore of support
It is 8%-11%.
3. application according to claim 1, it is characterised in that the concentration of the bone-inducing activity cell factor is 0.1-1mg/
cm3, the concentration of the sustained release agent is 0.2-2%.
4. application according to claim 1, it is characterised in that the concentration of the bone-inducing activity cell factor is 0.6-1mg/
cm3, the concentration of the sustained release agent is 0.2%.
5. application according to claim 1, it is characterised in that the concentration of the bone-inducing activity cell factor is 0.6-1mg/
cm3, the concentration of the sustained release agent is 1-2%.
6. the application according to one of claim 1 to 5, it is characterised in that method of the artificial bone to comprise the steps
Prepare：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；Cleaning；Baking oven is put into after cleaning up at 50-100 DEG C
It is dried 3-4 hours；Then, by coral and (NH4)2HPO4The aqueous solution be together put into autoclave, (NH4)2HPO4The aqueous solution
Mass concentration be 0.05-1mol/L, (NH4)2HPO4(NH in solution4)2HPO4With with CaCO3The mol ratio of the coral of metering
It is 1:10-15；Again to (NH4)2HPO4Potassium dihydrogen phosphate, per liter of (NH are added in solution4)2HPO4Potassium dihydrogen phosphate is added in solution
1mg；Reaction 12 hours, afterwards deionized water boil 4 hours, change deionized water once per half an hour, remove inorganic ions；Again
Gamma-rays illumination-based disinfection is used, degradable coral hydroxyapatite support is obtained；
(2) recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent are configured to the aqueous solution being well mixed；
(3) recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent are attached to the surface of degradable coral hydroxyapatite support
And in hole.
7. application according to claim 6, it is characterised in that methods described comprises the steps：
(1) degradable coral hydroxyapatite is prepared by coral
Taking coral carries out cutting polishing, is prepared into required size, shape；Cleaning；Baking oven is put into after cleaning up at 50-100 DEG C
It is dried 3-4 hours；Then, by coral and (NH4)2HPO4The aqueous solution be together put into autoclave, (NH4)2HPO4The aqueous solution
Mass concentration be 0.05-1mol/L, (NH4)2HPO4(NH in solution4)2HPO4With with CaCO3The mol ratio of the coral of metering
It is 1:10-1:15；Again to (NH4)2HPO4Potassium dihydrogen phosphate, per liter of (NH are added in solution4)2HPO4Biphosphate is added in solution
Potassium 1mg；Reaction 12 hours, afterwards deionized water boil 4 hours, change deionized water once per half an hour, remove inorganic ions；
Gamma-rays illumination-based disinfection is used again, degradable coral hydroxyapatite support is obtained, finally by degradable coral hydroxyapatite
Frame is dried packing cillin bottle；
(2) preparation of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent
Recombinant human bone morphorgenetic protein-2 mature peptide is configured to the aqueous solution of concentration 0.1-1mg/ml, adds in aqueous solution and accounts for
The sustained release agent of aqueous solution gross weight 0.2%-2%, stirring at low speed is abundant to mixing under low temperature, obtains recombinant human bone morphorgenetic protein-2
The homogeneous mixture solotion of mature peptide and sustained release agent；
(3) recombinant human bone morphorgenetic protein-2 mature peptide is attached on degradable coral hydroxyapatite support with sustained release agent, will be walked
Suddenly the mixed solution that (2) obtain is added in the degradable coral hydroxyapatite support that step (1) is obtained, and addition is 0.1-
1mg/cm3, low-temperature negative-pressure makes the mixed solution of recombinant human bone morphorgenetic protein-2 mature peptide and sustained release agent well into degradable coral
In the hole of coral hydroxyapatite scaffold, less than -30 DEG C vacuumize, freeze-drying, obtain described artificial bone, and artificial bone is close
Envelope, -20 DEG C long-term preserve.
8. application according to claim 7, it is characterised in that the pressure in the step (1) in reactor is：4-
9MPa, temperature is：270-280℃.
9. application according to claim 7, it is characterised in that with CaCO in the step (1)3The coral of metering with
(NH4)2HPO4Ratio be：10:1, the pressure in reactor is：7MP, temperature is：280℃.
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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 |
| 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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