CN101584884A - Method for preparing biomimetic artificial bone materials for biodegradable tissue engineering - Google Patents
Method for preparing biomimetic artificial bone materials for biodegradable tissue engineering Download PDFInfo
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Abstract
The invention discloses a method for preparing biomimetic artificial bone materials for biodegradable tissue engineering. The method comprises the following steps: adding a hydroxyapatite suspension to a human-like collagen solution; adding Genipin for crosslinking; performing vacuum freeze-drying to obtain human-like collagen/ hydroxyapatite powder; adding the human-like collagen/ hydroxyapatite powder to a chitosan solution; using an NaOH solution to adjust the pH of the solution to 7.0; repeatedly adding the human-like collagen solution and the chitosan solution; adding a Genipin solution for crosslinking; freeze-drying a mixed solution; inoculating the obtained material with BMSCs of which the cell density is 5*10<4> cells/cm<2>; performing culture for 1 to 10 days; using an induction medium to culture BMSCs cells so as to induce the BMSCs cells to differentiate towards an osteogenesis direction; and forming bone repair composite materials with bone inducing activity. The artificial bone materials prepared by the method are excellent in bone inducing activity and immune compatibility, thereby thoroughly putting an end to inevitable viral hidden trouble of animal collagen scaffold materials and greatly improving use safety.
Description
Technical field
The present invention relates to a kind of method for preparing biomimetic artificial bone materials for biodegradable tissue engineering, belong to biomedical materials field.
Background technology
Whether excellent biocompatibility be to weigh one of bone renovating material important indicator.Because natural biologic material has excellent biological compatibility, Recent study person is applied to it in bone renovating material or the bone tissue engineer more, especially animal collagen.But animal collagen extracts from animal tissue, can't eliminate as viral hidden danger such as bovine spongiform encephalopathy, swine fever epidemic disease, bird flus, also can't eliminate hepatitis, acquired immune deficiency syndrome (AIDS) etc. in people's collagen (from people's Placenta Hominis, bone etc.) leaching process infects dangerous in the same old way, therefore, animal sources type or people source collagen type are subjected to the restriction of World Health Organization (WHO) in application.Moreover the collagen protein of animal sources type and people's tissue extraction collagen are the insoluble type of water, can only be dissolved in acid or alkali, no matter used reagent is acid or alkali all can produce cytotoxicity, cell adhesion is poor, and soda acid is difficult for removing after the molding, and these shortcomings are restricted its potential purposes exploitation.
And Human-like Collagen II is a kind of high-molecular biologic albumen of producing by technique for gene engineering, its biology performance is than animal sources collagen protein excellence, has the good cell adhesion, short new cell forms and urgees epithelial cell and forms that (1.4-1.6 that 48 hours short epithelial cell formation speed of Human-like Collagen is the normal control group doubly, animal body collagen then is 1.05-1.1 times) function, and its good processability, virus-free hidden danger, good water solubility (cytotoxicity of having avoided the soda acid dissolvent residual to bring), rejection is low (because this Human-like Collagen is gene constructed by human collagen, therefore, after entering human body, its immune rejection significantly reduces with comparing from the collagen protein of animal tissue).So, in order to improve the biocompatibility of further raising bone renovating material, with the animal collagen in the alternative traditional bone renovating material of Human-like Collagen II.
For mechanical strength and the realization controlled degradation that improves bone renovating material, the researcheres glutaraldehydes that adopt as cross-linking agent more.Because the glutaraldehyde pair cell has big toxicity, and after implanting with its crosslinked bone renovating material, its catabolite also can cause certain injury to body tissue.Therefore, adopt the crosslinked Human-like Collagen II of natural biological cross-linking agent genipin bone composite material in this research.Genipin is a kind of natural biological cross-linking agent that is extracted from the cape jasmine fruit, belongs to the chemical compound of iridoids, has a plurality of active groups such as hydroxyl, carboxyl, can carry out crosslinked with unimolecule and polymolecular form.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing biodegradable tissue engineering with artificial bone, this method technology is simple, prepared artificial bone bone-inducting active and immune compatibility excellence, thoroughly stopped the inevitable viral hidden danger of animal collagen timbering material, safety in utilization increases substantially.
Implementation procedure of the present invention is as follows:
A kind of method for preparing biodegradable used in tissue engineering biomimetic artificial bone materials is characterized in that may further comprise the steps:
1) Human-like Collagen is become 1%～5% solution with dissolved in distilled water;
2) preparation hydroxyapatite suspension;
3) the hydroxyapatite suspension is added in the Human-like Collagen solution, gentle agitation 6h, standing over night, transfer to pH value of solution to 7.0 with 0.1MNaOH, the 4 ℃ of crosslinked 1d of genipin that add 0.1-1%, vacuum lyophilization 24h makes Human-like Collagen (RHLCII)/hydroxyapatite (nHA) powder;
4) chitosan is dissolved into 0.01%～0.04% solution with diluted acid;
5) Human-like Collagen/hydroxyapatite powder is added in the chitosan solution, transfer to pH value of solution to 7.0 with 0.1MNaOH solution;
Repeat (repeating 2-5 time) following steps: the Human-like Collagen solution of adding 1%～5% is 6.4 to pH, and adding 0.01%～0.04% chitosan solution is adjusted pH value of solution to 7.0 with NaOH solution;
Under ice bath, utilize ultrasonic wave concussion that solution is fully disperseed, add the crosslinked 1-3d of genipin solution of 0.1-1% then;
6) with in the above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h, precooling 3h final vacuum lyophilization 48h, the PBS buffer of reuse pH 7.3 embathes repeatedly, then with preservation after its secondary lyophilizing;
7) be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm;
8) behind the cultivation 1-10d, change inducing culture and cultivate the BMSCs cell, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
In the above-mentioned steps, a kind of people source collagen type of used Human-like Collagen for using the gene recombined escherichia coli high density fermentation to produce.
Above-mentioned steps 2) in, the nanometer hydroxyapatite preparation method is as follows: Ca (NO
3)
2Solution and (NH
4)
3PO
4Solution reaction, the pH value NH of reaction system
3H
2O regulates and makes it keep pH9 ± 0.1, and equation is as follows.
Ca(NO
3)
2·4H
2O+(NH
4)
3PO
4·3H
2O→Ca
10(PO
4)
6(OH)
2
In the above-mentioned steps, used diluted acid is any one in phosphoric acid,diluted, spirit of vinegar, dilute formic acid or the rare propanoic acid.
Human-like Collagen of the present invention and nanometer hydroxyapatite mass ratio are (0.25～4): 1, and used chitosan molecule amount is 400,000～600,000Da, the amount of chitosan solution accounts for the 1wt%～4wt% of whole solution;
Step 3), 5) genipin aqueous solution shared in accounts for 0.5%～1% of mixed solution.
Above-mentioned steps 6) in, used cylindrical mold diameter is 10mm, height 10～30mm.
Above-mentioned steps 6) in, precooling temperature is in-20 ℃～-196 ℃ scopes.
In order better to control the structure and the performance of bone renovating bracket material, the present invention adopts Minitab statistics software that the bone renovating material moulding process is optimized, and set up the structure that response surface model instructs bone renovating material, shortened the technology of preparing cycle greatly, reduced workload, produced for the later stage industrialization and established certain basis.
The specific embodiment
The invention will be further described below by concrete embodiment, and percentage ratio of the present invention is mass percent.
Embodiment 1:
Under 100 ℃, Ca (NO
3)
2Solution and (NH
4)
3PO
4Solution reaction, the pH value of reaction system 0.1M NH
3H
2O regulates and makes it keep pH9 ± 0.1, continues stirring reaction 4h, prepares acicular nanometer hydroxyapatite suspension.Reaction equation is as follows:
Ca(NO
3)
2·4H
2O+(NH
4)
3PO
4·3H
2O→Ca
10(PO
4)
6(OH)
2
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 6: 4, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with spirit of vinegar; Dropwise add the RHLCII/nHA powder in the chitosan solution, adjust pH value of solution to 7.0 with 0.1MNaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH, above step triplicate, the adding total amount of chitosan solution is 1% of a mixed solution quality, under ice bath, utilize ultrasonic wave concussion that mixed solution is fully disperseed, add the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 7d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 2:
Human-like Collagen become 3% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 6: 4, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan become 0.01% solution with acetate dissolution; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step repeats four times, and the adding total amount of chitosan is 2% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 4d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 3:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 6: 4, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with dilute formic acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1MNaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 3% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 8d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 4:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 4: 4, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with diluted acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 4% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 3d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 5:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 2: 8, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with diluted acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 2% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.52% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 5d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 6:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 4: 6, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with diluted acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 2% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 6d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 7:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 7: 2, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1M NaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.01% solution with diluted acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 2% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 7d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Embodiment 8:
Human-like Collagen become 4% solution with dissolved in distilled water, the nanometer hydroxyapatite suspension of aforementioned preparation is dropwise added in the Human-like Collagen solution, making Human-like Collagen and nanometer hydroxyapatite mass ratio is 6: 4, gentle agitation 6h, standing over night is adjusted pH value of solution to 7.0 with 0.1MNaOH.Add the crosslinked 1d of genipin, vacuum lyophilization 24h makes recombination human collagen I I (RHLCII)/nanometer hydroxyapatite (nHA) powder.Chitosan is dissolved into 0.02% solution with diluted acid; The RHLCII/nHA powder is dropwise added in the chitosan solution, adjusts pH value of solution to 7.0 with 0.1M NaOH, add once more collagen to pH be 6.4, add aforementioned chitosan solution once more, adjust pH value of solution to 7.0 with NaOH.Above step triplicate, the adding total amount of chitosan is 2% of a mixed solution quality, utilizes ultrasonic wave concussion that mixed solution is fully disperseed under ice bath, adds the crosslinked 1-3d of genipin solution then.The final genipin solution that keeps accounts for 0.61% of total solution.With above-mentioned mixed solution impouring stainless steel mould, at 4 ℃ of following cold preservation 4h ,-80 ℃ of precooling 3h final vacuum lyophilization 48h.Reuse PBS (pH=7.3) buffer embathes repeatedly, then with preservation after its secondary lyophilizing; Cobalt
60After the sterilization, be 5 * 10 with cell density
4Cells/cm
2BMSCs to be inoculated in the length of side be 5mm, thickness is on the above-mentioned material of 2mm; Change the BMSCs cell on the inducing culture culture materials behind the cultivation 8d, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
Bone-repairing composite material performance test experiment
1, the mechanical performance of timbering material
The comprcssive strength performance that various bones are repaired compound support frame material detects (pure nHA is contrast) by Instron 5565 type electronic universal experimental machine.Sample is made disk: Ф 10mm * 13mm, adopt the 100N load transducer, the velocity of displacement of crossbeam is 1mm/min, the stress and strain when obtaining sample breakage also calculates the Young's modulus of material.5 samples of every kind of parallel detection of material.With Human-like Collagen and nanometer hydroxyapatite mixing quality ratio is that 4: 6 timbering material is an experiment material, test result shows: this support has the favorable mechanical performance, sample comprcssive strength is 80.46 ± 5.4MPa, strain (%) is 50 ± 1.2, and the comprcssive strength of nHA and Young's modulus all can't be measured because of too crisp.
2, the external degradation of timbering material experiment
(it is 7 simulated body fluid SBF solution that Ф 10mm * 13mm) puts into pH value, and in 37 ℃ constant temperature shaking table, low speed shakes, and takes out every 7-10d, is put in the baking oven 40 ℃ of dryings, weighs with all samples.The mass loss rate of sample is in the swelling process:
D＝(m1-m2)/m1×100％
In the formula: m1 is the preceding quality of degraded; M2 is the quality after degrading;
The result shows be increased to 2% o'clock of chitosan amount, and the comprcssive strength of material is higher, mass loss rate is lower after 3 months, has remedied the shortcoming of hydrophilic recombination human collagen of single usefulness and degradation of chitosan excessive velocities, satisfies the requirement that the bone reparation is reached.
3, Study on biocompatibility
When Human-like Collagen, nanometer hydroxyapatite and chitosan compound tense, can promote sticking and propagation of mesenchymal stem cells MSCs better, space structure and the environment that is more suitable for its growth is provided for mesenchymal stem cells MSCs.
4, osteogenic induction activity research
All samples is through BMSCs cell (complete medium is contrast) behind inducing culture, viable cell density is doubly growth, cultivating the no dead cell in back in 14 days occurs, the attaching rate is near 100%, the ALP value is 570.4 (pNPP mM/min)/(μ gDNA), activity is significantly higher than activity 57.8 (pNPPmM/min)/(the μ gDNA) in the culture dish, almost is 10 times in the culture dish.Suitable with CPC material activity by U.S. FDA standard A LP value, and have the calcium tuberosity to produce.
Claims (9)
1. method for preparing biomimetic artificial bone materials for biodegradable tissue engineering is characterized in that may further comprise the steps:
1) Human-like Collagen is become 1%～5% solution with dissolved in distilled water;
2) preparation hydroxyapatite suspension;
3) the hydroxyapatite suspension is added in the Human-like Collagen solution, transfer to pH value of solution to 7.0 with NaOH solution, 4 ℃ of genipin that add 0.1-1% are crosslinked, and vacuum lyophilization makes Human-like Collagen/hydroxyapatite powder;
4) chitosan is dissolved into 0.01%～0.04% solution with diluted acid;
5) Human-like Collagen/hydroxyapatite powder is added in the chitosan solution, transfer to pH value of solution to 7.0 with NaOH solution, repeat to add 1%～5% Human-like Collagen solution and 0.01%～0.04% chitosan solution, add the crosslinked 1-3d of genipin solution of 0.1-1% then;
6) with above-mentioned mixed solution vacuum lyophilization;
7) be 5 * 10 with cell density
4Cells/cm
2BMSCs be inoculated on the step 6) material;
8) behind the cultivation 1-10d, change inducing culture and cultivate the BMSCs cell, induce it to the differentiation of skeletonization direction, final formation has the bone-repairing composite material of inducing bone active.
2. according to the described method for preparing biomimetic artificial bone materials for biodegradable tissue engineering of claim 1, it is characterized in that: a kind of people source collagen type of used Human-like Collagen for using the gene recombined escherichia coli high density fermentation to produce.
3. according to the described method for preparing biomimetic artificial bone materials for biodegradable tissue engineering of claim 1, it is characterized in that the hydroxyapatite suspension uses following method preparation: Ca (NO
3)
2Solution and (NH
4)
3PO
4Solution reaction, the pH value NH of reaction system
3H
2O regulates and makes it keep pH 9 ± 0.1.
4. according to the method for the biodegradable used in tissue engineering biomimetic artificial bone materials of the described preparation of claim 1, it is characterized in that: used chitosan molecule amount is 400,000～600,000Da.
5. according to the described method for preparing biomimetic artificial bone materials for biodegradable tissue engineering of claim 1, it is characterized in that: the total amount of chitosan solution accounts for 1%～4% of whole solution.
6. according to the described method for preparing biomimetic artificial bone materials for biodegradable tissue engineering of claim 1, it is characterized in that: described diluted acid be in phosphoric acid,diluted, spirit of vinegar, dilute formic acid or the rare propanoic acid any one.
7. according to the described method for preparing biomimetic artificial bone materials for biodegradable tissue engineering of claim 1, it is characterized in that: Human-like Collagen and nanometer hydroxyapatite mass ratio are (0.25～4): 1.
8. according to the method for the biodegradable used in tissue engineering biomimetic artificial bone materials of the described preparation of claim 1, it is characterized in that: genipin aqueous solution used step 3) and 5) accounts for 0.5%～1% of mixed solution.
9. according to the method for the biodegradable used in tissue engineering biomimetic artificial bone materials of the described preparation of claim 1, it is characterized in that: in the step 6), precooling temperature is at-20 ℃～-196 ℃.
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