CN104984393B - A kind of bone tissue engineering stent material and preparation method thereof - Google Patents
A kind of bone tissue engineering stent material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of bone tissue engineering stent material and preparation method thereof, fibroin/hydroxyapatite powder is synthesized according to bionic principle first, is dissolved in sericin solution, then by sericin solution（Containing fibroin/hydroxyapatite）, chitosan solution, the mixing of phosphoglycerol sodium solution, then mixed solution by temperature control is changed into gel, freezes gel is pre-cooled, thaws, freezes again, and final freeze-drying, produce bone tissue engineering stent material.The tissue engineering bracket material has excellent in mechanical performance, good biocompatibility, the degradability matched with bone cell growth, good moldability, and the advantage that osteocyte can be induced to grow into material.The timbering material can accelerate the physiology course that osteocyte is grown into, and with bone uptake is promoted, can shorten the rehabilitation duration of patient, accelerate the process of clinical treatment, reduce the pain of patient.Preparation method has reaction condition gentle, and technique is simple, with low cost, the advantage suitable for amplification production.
Description
Technical field
The present invention relates to tissue engineering material, more particularly to a kind of bone tissue engineering stent material and preparation method thereof, category
In new high-tech material manufacturing technology field.
Background technology
The treatment of Cranial defect is one of problem on Orthopedic Clinical all the time.The patient of global annual fracture and Cranial defect
Number is in terms of necessarily, and traditional treatment method mainly has autologous bone transplanting, homogeneous allogenic bone transplantation, an artificial bone graft, but these
All there is larger defect in method.Autologous bone transplanting exists as " goldstandard " of bone graft technique and causes new disease damage, source
It is limited, cause the defects such as the infection of bone area, pain, operating time length；Homogeneous allogenic bone transplantation, which exists, to be easily absorbed by the body, susceptible
Contaminate, immunological rejection is serious etc.；There is limited source in artificial bone graft, skeletonization is difficult, the defect such as the variation of raw material porosity is big.
Due to many disadvantages of existing bone defect healing method, current researchers are by repairing bone defect target diversion bone tissue engineer.
Bone tissue engineer is the principle and method using organizational engineering, develops the bone with repairing bone defect ability and substitutes
The a science of thing, general principle is to construct timbering material-seed cell-growth factor complex in vitro, is then transplanted to
At Cranial defect, absorption and the hyperplasia of tissue by material realize that itself bone tissue replaces tissue engineered bone, finally realize bone
Form and reparation functionally.The research and application of bone tissue engineer will overcome autologous bone transplanting in existing bone defect healing
The problem of source is few, allogenic bone transplantation has rejection and deficiency.Therefore by studying bone tissue engineer come repairing bone defect, no
It is only significant to China's bone-specific drug field, and there is impetus to the development of China's bone tissue engineer.
The key link of bone tissue engineer research is that development can be as cell transplantation and the support material of guiding new bone growth
Material, using the substitute as extracellular matrix.Current timbering material mainly has artificial synthesized timbering material and natural scaffold materials.
Artificial synthesized timbering material is the extracellular matrix substitute synthesized with physicochemical method, and main inorganic material has hydroxyl
Apatite, tricalcium phosphate；High-molecular organic material has PLA, polyglycolic acid etc..Zhao etc. uses the side of biomimetic mineralization
Method, makes PLGA mesh inwall deposit apatite mineralized layer, and the method significantly improves PLGA and the early stage of bone marrow stroma stem cell is sticked
It is attached, or can clinically shorten the treatment time of patient.Hydroxyapatite and polyamide (n-HA/PA66) are combined by Zhou Liwei etc.
Into artificial bone, and the defect of rabbit headbone is successfully repaired, the good biocompatibility of composite material exhibits in experiment, not to encephalic
Metabolism produces harmful effect, blur boundary between material and skull after being implanted into 12 weeks, shows the bone guided effect of composite.
Natural scaffold materials mainly have natural macromolecular material（Chitosan, chitin, hyaluronic acid, collagen etc.）With it is natural
Inorganic material（Natural coral, de- albumen bone of human or animal etc.）.The coral hydroxyapatite in vitro culture marrow base such as Cao Gang
Cell plastid, it is found that cell can continue to breed and express osteogenic activity in vitro, Amaral etc. arrives murine stromal cells plantation
In vitro culture discovery is carried out on chitosan film, cell can largely stick, expand and have skeletonization tendency.The coral support such as Liu
Compound autologous fat stem cell, successfully repairs the severe full-thickness defects of dog socket of the eye wall.But CHA bad mechanical properties, it is impossible to be used in hold
, there is certain volume forfeiture at weight position, it is difficult to moulding, infiltration rate is excessively slow during bulk bone grafting, and degradation speed is difficult to regulate and control after implantation
It can not ignore etc. defect.
It may be said that all kinds of supports developed so far all there are problems that.Its great advantage of artificial synthesized timbering material
It is wide in variety, selectable range is wide, and biodegradation rate is adjustable.In addition, synthetic material also has mechanical property and good processability,
It is cheap, favorable reproducibility, beneficial to large-scale production.Have the disadvantage that the compatibility ratio natural material of biocompatibility and cell is poor.
Natural scaffold materials typically have cell signal recognition capability, can promote adhesion, propagation and the differentiation of cell, mostly
Have no toxic side effect, with good biocompatibility and good biological degradability, show that synthetic material is incomparable
Advantage.But its processability, reappearance, degradation speed adjustability are not satisfactory.Partly natural-synthetic material overcomes some and asked
Topic, but also annoying Tissue Engineering Study personnel the problem of existing in terms of shaping, degraded.Especially such as human body soft tissue
Support, to mould molding, strength, elongation, elasticity, pliability, and the degradation property matched with cell growth etc.
It is required that very high.
Therefore, develop it is a kind of there is induction osteocyte to grow into, degrade also controllable, with outstanding mechanical property and biofacies
The problem of bone tissue tissue engineering bracket of capacitive is urgent need to resolve.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide a kind of bone tissue engineering stent material,
The tissue engineering bracket material has an excellent in mechanical performance, good biocompatibility, the degradability matched with bone cell growth, into
Type is good, and the advantage that osteocyte can be induced to grow into material, and the timbering material accelerates the physiology course that osteocyte is grown into, and has
Promote bone uptake, the rehabilitation duration of patient can be shortened, accelerate the process of clinical treatment, reduce the pain of patient.
It is a further object to provide a kind of preparation method of described bone tissue engineering stent material, the preparation
Method has reaction condition gentle, and technique is simple, with low cost, the advantage suitable for amplification production.
To realize the object of the invention, following technical scheme is employed：
A kind of bone tissue engineering stent material preparation method, step is as follows,
1）The preparation of solution：
Silk fibroin powder is dissolved in deionized water, the silk fibroin protein solution that concentration is 8-15mg/ml is made into；
Phosphoric acid solution is diluted with deionized water, the phosphoric acid solution that concentration is 0.01-0.1mol/L is made into；
Calcium hydroxide powder is dissolved in deionized water, the aqua calcis that concentration is 0.01-0.05mol/L is made into；
Sericin powder is dissolved in deionized water, the sericin solution that concentration is 4%-8% (W/V) is made into；
Dissolve the chitosan in dilute acid soln, be made into the chitosan solution that concentration is 2-3% (W/V)；
Sodium glycero-phosphate is dissolved in deionized water, concentration is made into for 50-60%（W/V）Phosphoglycerol sodium solution；
2）Prepare fibroin/hydroxyapatite powder：By the 1st）The silk fibroin protein solution that step prepares uniformly is mixed with phosphoric acid solution
Close；Then in 60-80 DEG C of water bath with thermostatic control, by the mixed liquor of fibroin albumen and phosphoric acid with the slow-paced of 40-60 drops/min
It is added dropwise in aqua calcis, after adding, 2-3h is stirred with 150-220r/min, stirring adjusts reacting solution pH value after terminating
For 9-10, the precipitation 24-48h in reaction temperature water-bath, vacuum filtration, with deionized water rinse at least three times, that is, obtain fibroin/
Hydroxyapatite powder；Wherein, the volume ratio of silk fibroin protein solution and phosphoric acid solution, aqua calcis is 1:（1.5-2）:
（10-15）；
3）By step 2）In obtained fibroin/hydroxyapatite powder be added to step 1）In the sericin solution of preparation
It is uniformly mixed, obtained silk gum/(fibroin/hydroxyapatite) suspension, wherein sericin and fibroin/hydroxyapatite
The mass ratio of powder is 1:0.5-1；
4）By step 3）Obtained silk gum/(fibroin/hydroxyapatite) suspension, is slowly added into chitosan solution
In, stir, the temperature of adjustment system is 1-10 DEG C, then step 1 is added dropwise）The phosphoglycerol sodium solution stirring of preparation
30min, obtains mixed solution；Wherein silk gum/(fibroin/hydroxyapatite) suspension, chitosan solution, phosphoglycerol sodium solution
Volume ratio be（0.67-1）:（5-6）:1；
5）By the 4th）Walk obtained mixed solution to pour into mould, mould is put into 37-38 DEG C of water bath with thermostatic control, makes mixing
Molten-the gel conversion of solution completion, precooling 2-5h at a temperature of gel rubber material is firstly placed on into -10--20 DEG C, defrosting 1-3h, then
6-10h is freezed in the refrigerator for going to -70--80 DEG C, freeze drier is then placed in and dries more than 24h, vacuum degree control is in 10-
50 Kpa, produce bone tissue engineering stent material.
Step 1）Described in the preparation method of silk fibroin powder be：Using the molten enzymolysis process of salt, by useless light defective cocoon shell or cocoon
Silk is boiled through ethers, alcohol organic solvent immersion to remove waxy substance therein, carbohydrate and ash impurities, then through water successively
Freeze-drying obtains micron order silk fibroin powder after boiling degumming salt is molten.
Step 1）The preparation method of middle sericin powder is：Cocoon shell is soaked into 24-48h respectively successively through ethers, alcohols,
Cleaned, dried, to remove wax and partial organic substances and impurity with distilled water；Cocoon shell distilled water is boiled at least 6h again, will
Sericin is dissolved in distilled water, and suction filtration obtains sericin solution, then produces the concentration of sericin solution, freeze-drying
Sericin powder.
Step 1）In diluted acid be watery hydrochloric acid or spirit of vinegar, concentration is 0.05-0.2mol/L.
Compared with prior art, the present invention has advantages below：
（1）Hydroxyapatite is sunk according to bionic principle and gathered on fibroin albumen, having synthesized fibroin/hydroxyapatite, will
Fibroin/hydroxyapatite is incorporated into silk gum/chitosan/sodium glycero-phosphate gel rubber system, prepares bone tissue engineering scaffold material
Material, while assigning the timbering material function that induction osteocyte is grown into, the osteoinductive of the timbering material is come from bionical human body
Collagen/hydroxyapatite, therefore lure bone uptake more excellent.
（2）Sericin (fibroin/hydroxyapatite)/chitosan/sodium glycero-phosphate organizational project branch obtained by the present invention
Frame material, possesses good biocompatibility, degraded Modulatory character and preferable mouldability, can be as treating Cranial defect
Tissue engineering bracket material.
（3）Sericin (fibroin/hydroxyapatite)/chitosan/sodium glycero-phosphate organizational project branch obtained by the present invention
Frame material changes into gel in any mould from solution, pre-cooled jelly, defrosting, freezes again, and final freeze-drying,
During this, the tissue engineering bracket material of excellent in mechanical performance is formed.And its mechanical property can add silk by adjusting
Amount and sericin solution, the ratio between chitosan solution and phosphoglycerol sodium solution of element/hydroxyapatite are regulated and controled.
（4）The preparation method reaction condition of the present invention is gentle, and technique is simple, low raw-material cost, suitable for amplification production；And
And material proportion can be adjusted as needed to reconcile material mechanical performance and degradation time etc., it is met the group of respective performances
Knit engineering scaffold material.
Brief description of the drawings
Fig. 1 is the external morphology figure of tissue engineering bracket material prepared by embodiment 1.
Fig. 2 is embodiment 2 and control group（Silk gum/fibroin/chitosan/sodium glycero-phosphate）The tissue engineering bracket material of preparation
The scanning electron microscope (SEM) photograph of material.Wherein Fig. 2 (A) and Fig. 2 (B) is the scanning electron microscope (SEM) photograph of embodiment 2, and Fig. 2 (C) is control group figure.
Fig. 3 is tissue engineering bracket material porosity test result curve map prepared by embodiment 2.
Fig. 4 is the dissolve-loss ratio test result curve map of tissue engineering bracket material prepared by embodiment 2.
Fig. 5 be embodiment it is 2-in-1 into fibroin/hydroxyapatite X-ray diffractogram.
Fig. 6 is bone tissue engineering stent material and control group prepared by embodiment 2（Pure ha）Infrared spectrum
Figure.Curve a is pure ha, and curve b is bone tissue engineering stent material prepared by embodiment 2.
Embodiment
Bone tissue engineering stent material preparation method of the present invention, is comprised the following steps that,
1）The preparation of the various solution needed：
Silk fibroin powder is dissolved in deionized water, the silk fibroin protein solution that concentration is 8-15mg/ml is made into；
Phosphoric acid solution is diluted with deionized water, the phosphoric acid solution that concentration is 0.01-0.1mol/L is made into；
Calcium hydroxide powder is dissolved in deionized water, the aqua calcis that concentration is 0.01-0.05mol/L is made into；
Sericin powder is dissolved in deionized water, the sericin solution that concentration is 4%-8% (W/V) is made into；
Dissolve the chitosan in dilute acid soln, be made into the chitosan solution that concentration is 2-3% (W/V)；Diluted acid is watery hydrochloric acid
Or spirit of vinegar, concentration is 0.05-0.2mol/L；
Sodium glycero-phosphate is dissolved in deionized water, concentration is made into for 50-60%（W/V）Phosphoglycerol sodium solution；
Wherein, silk fibroin powder is prepared as follows obtaining：Using the molten enzymolysis process of salt, by useless light defective cocoon shell or silk
Boiled successively through ethers, alcohol organic solvent immersion to remove waxy substance therein, carbohydrate and ash impurities, then through water
Freeze-drying obtains micron order silk fibroin powder after degumming salt is molten.
Wherein, sericin powder is prepared as follows obtaining：Cocoon shell is soaked into 24- respectively successively through ethers, alcohols
48h, is cleaned with distilled water, dried, to remove wax and partial organic substances and impurity；Cocoon shell distilled water is boiled at least again
6h, sericin is dissolved in distilled water, and suction filtration obtains sericin solution, then that the concentration of sericin solution, freezing is dry
It is dry to produce sericin powder.
2）Prepare fibroin/hydroxyapatite powder：By the 1st）The silk fibroin protein solution that step prepares uniformly is mixed with phosphoric acid solution
Close；Then in 60-80 DEG C of water bath with thermostatic control, by the mixed liquor of fibroin albumen and phosphoric acid with the slow-paced of 40-60 drops/min
It is added dropwise in aqua calcis, after adding, 2-3h is stirred with 150-220r/min, stirring adjusts reacting solution pH value after terminating
For 9-10, the precipitation 24-48h in reaction temperature water-bath, vacuum filtration, with deionized water rinse at least three times, that is, obtain fibroin/
Hydroxyapatite powder；Wherein, the volume ratio of silk fibroin protein solution and phosphoric acid solution, aqua calcis is 1:（1.5-2）:
（10-15）.
3）By step 2）In obtained fibroin/hydroxyapatite powder be added to step 1）In the sericin solution of preparation
It is uniformly mixed, obtained silk gum/(fibroin/hydroxyapatite) suspension, wherein sericin and fibroin/hydroxyapatite
The mass ratio of powder is 1:（0.5-1）；
4）By step 3）Obtained silk gum/(fibroin/hydroxyapatite) suspension, is slowly added into chitosan solution
In, stir, the temperature of adjustment system is 1-10 DEG C, then step 1 is added dropwise）The phosphoglycerol sodium solution stirring of preparation
30min, obtains mixed solution；Wherein silk gum/(fibroin/hydroxyapatite) suspension, chitosan solution, phosphoglycerol sodium solution
Volume ratio be（0.67-1）：（5-6）：1.
5）By the 4th）Walk obtained mixed solution to pour into mould, mould is put into 37 ± 5 DEG C of waters bath with thermostatic control, makes mixing
Molten-the gel conversion of solution completion, precooling 2-5h at a temperature of gel rubber material is firstly placed on into -10--20 DEG C, defrosting 1-3h, then
6-10h is freezed in the refrigerator for going to -70--80 DEG C, freeze drier is then placed in and dries more than 24h, vacuum degree control is in 10-
50 Kpa, produce bone tissue engineering stent material.
The bone tissue engineering stent material that the present invention is obtained, is a kind of hard tissue engineering support, with being adapted to bone tissue
Mechanical property and the degradability that matches with bone cell growth, osteocyte can be induced to grow into wherein, and with promoting cell life
Long property.The present invention can be by adjusting the ratio between system each component, or changes the support that process conditions can control to obtain
The mechanical property and osteoinductive of material.
The present invention is described in further details with specific embodiment below in conjunction with the accompanying drawings.
, it is necessary to first prepare sericin powder, silk fibroin powder and fibroin/hydroxyl before specific embodiment preparation is carried out
Base apatite powder.
Sericin powder used is prepared using following methods：
Cocoon shell is soaked into 48h through ethers, alcohols respectively successively, is cleaned, dried with distilled water, is had to remove wax and part
Machine thing and impurity；Cocoon shell distilled water is boiled at least 6h again, sericin is dissolved in distilled water, suction filtration obtains sericin
Solution, then obtains sericin powder by the concentration of sericin solution, freeze-drying.
Micron silk fibroin powder used is prepared using following methods：
Cocoon shell is first removed into wax, organic matter and impurity through ethers, alcohols immersion, degumming is boiled and obtains fibrous fibroin egg
In vain.Fibroin fiber is dissolved in CaC12/C2H5OH/H2In O organic-inorganic mixed liquors, dialysis 3d takes out, and with membrane filtration and rushes
Silk fibroin protein solution is obtained after washing, is finally concentrated silk fibroin protein solution, freeze drying obtains a micron silk fibroin powder.
Fibroin/hydroxyapatite powder used is prepared using following methods：
11.8mg/ml silk fibroin protein solution 21.8ml is uniformly mixed with 0.087mol/L phosphoric acid solutions 41.5ml.So
Afterwards in 70 DEG C of waters bath with thermostatic control, mixed liquor is added dropwise in 300ml aqua calcises with the slow-paced of 60 drops/min, plus
After complete, continue to stir 2-3h with (150-220r/min) speed, regulation reacting solution pH value is 9-10 after stirring terminates, in reaction
48h is placed in temperature water bath, is filtered by vacuum, is rinsed three times with deionized water, prepares fibroin/hydroxyapatite powder.Fig. 5
For the X-ray diffractogram of fibroin/hydroxyapatite powder of synthesis.
Embodiment 1：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the chitosan that concentration is 2% (W/V)
Solution；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.05g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtain
In 1.34ml sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 6 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min, and it is 7.0 to insert in mould and adjust pH mixed solution, and mould is put into 37.5 DEG C of bath temperatures,
About 15min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 2h in -20 DEG C of refrigerators, defrosting 1h, -77 DEG C are transferred to
8h is freezed in refrigerator, is transferred in freeze drier and dries 24h, vacuum is 30Kpa, you can obtain bone tissue engineering scaffold material
Material.Fig. 1 is the external morphology figure of tissue engineering bracket material prepared by embodiment 1.
Embodiment 2：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the chitosan that concentration is 2% (W/V)
Solution；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.05g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtained 1ml
In sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 10 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min；It is 7.0 to insert in mould and adjust pH mixed solution, and mould is put into 37.5 DEG C of bath temperatures,
About 20min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 2h in -20 DEG C of refrigerators, defrosting 1h, -80 DEG C are transferred to
8h is freezed in refrigerator, is transferred in freeze drier and dries 24h, vacuum is 25Kpa, you can obtain bone tissue engineering scaffold material
Material.Fig. 2 is implementation 2 and control group（Silk gum/fibroin/chitosan/sodium glycero-phosphate）The tissue engineering bracket material of preparation is swept
Retouch electron microscope.
Embodiment 3：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the chitosan that concentration is 2% (W/V)
Solution；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.075g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtain
In 1.34ml sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 8 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min；It is 7.0 to insert in mould and adjust pH mixed solution, mould is put into 37 DEG C of bath temperatures, about
10min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 2h in -20 DEG C of refrigerators, defrosting 2h, be transferred to -80 DEG C of ice
8h is freezed in case, is transferred in freeze drier and dries 24h, vacuum is 50Kpa, you can obtain bone tissue engineering scaffold material
Material.
Embodiment 4：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the shell that concentration is 2.5% (W/V) and gather
Sugar juice；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.05g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtain
In 1.34ml sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 7 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min；It is 7.0 to insert in mould and adjust pH mixed solution, mould is put into 37 DEG C of bath temperatures, about
15min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 3h in -20 DEG C of refrigerators, defrosting 2h, be transferred to -80 DEG C of ice
8h is freezed in case, is transferred in freeze drier and dries 24h, vacuum is 28Kpa, you can obtain bone tissue engineering scaffold material
Material.
Embodiment 5：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the chitosan that concentration is 2% (W/V)
Solution；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.05g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtain
In 1.5ml sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 4 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min；It is 7.0 to insert in mould and adjust pH mixed solution, and mould is put into 37.5 DEG C of bath temperatures,
About 20min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 4h in -20 DEG C of refrigerators, defrosting 2h, -80 DEG C are transferred to
8h is freezed in refrigerator, is transferred in freeze drier and dries 24h, vacuum is 40Kpa, you can obtain bone tissue engineering scaffold material
Material.
Embodiment 6：
A kind of preparation method of bone tissue engineering stent material, specifically includes following steps：
（1）Sericin powder is dissolved in deionized water, the sericin solution that concentration is 5.0% (W/V) is made into；
Dissolve the chitosan in the dilute hydrochloric acid solution that concentration is 0.1mol/L, be made into the chitosan that concentration is 2% (W/V)
Solution；
Sodium glycero-phosphate is dissolved in deionized water, it is 50% to be made into concentration（W/V）Phosphoglycerol sodium solution；
（2）0.05g fibroins/hydroxyapatite powder under room temperature condition, will be weighed and be added to step（1）In obtain
In 1.34ml sericin solution, stir；
（3）By step（2）In obtain silk gum/（Fibroin/hydroxyapatite）Mixed liquor is added in 10ml chitosans, stirs
Mix well mixed；Now the temperature of regulation and control mixed system is after 10 DEG C, then step to be added dropwise（1）Obtained sodium glycero-phosphate is molten
Liquid 2ml, stirs 30min；It is 7.0 to insert in mould and adjust pH mixed solution, mould is put into 37 DEG C of bath temperatures, about
15min can complete molten-gel conversion, and gel rubber material is put into pre-freeze 2h in -20 DEG C of refrigerators, defrosting 2h, be transferred to -77 DEG C of ice
8h is freezed in case, is transferred in freeze drier and dries 24h, vacuum is 24Kpa, you can obtain bone tissue engineering scaffold material
Material.
Finally illustrate, above-described embodiment is merely to illustrate technical scheme and unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to skill of the invention
Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this
Among the right of invention.
Claims (5)
1. a kind of bone tissue engineering stent material preparation method, it is characterised in that：Step is as follows,
1）The preparation of solution：
Silk fibroin powder is dissolved in deionized water, the silk fibroin protein solution that concentration is 8-15mg/ml is made into；
Phosphoric acid solution is diluted with deionized water, the phosphoric acid solution that concentration is 0.01-0.1mol/L is made into；
Calcium hydroxide powder is dissolved in deionized water, the aqua calcis that concentration is 0.01-0.05mol/L is made into；
Sericin powder is dissolved in deionized water, the sericin solution that concentration is 4%-8% (W/V) is made into；
Dissolve the chitosan in dilute acid soln, be made into the chitosan solution that concentration is 2-3% (W/V)；
Sodium glycero-phosphate is dissolved in deionized water, concentration is made into for 50-60%（W/V）Phosphoglycerol sodium solution；
2）Prepare fibroin/hydroxyapatite powder：By the 1st）The silk fibroin protein solution that step prepares uniformly is mixed with phosphoric acid solution；So
Afterwards in 60-80 DEG C of water bath with thermostatic control, the mixed liquor of fibroin albumen and phosphoric acid is added dropwise to 40-60 drops/the slow-paced of min
In aqua calcis, after adding, 2-3h is stirred with 150-220r/min, regulation reacting solution pH value is 9- after stirring terminates
10, the precipitation 24-48h in reaction temperature water-bath, vacuum filtration are rinsed at least three times with deionized water, that is, obtain fibroin/hydroxyl
Apatite powder；Wherein, the volume ratio of silk fibroin protein solution and phosphoric acid solution, aqua calcis is 1:（1.5-2）:（10-
15）；
3）By step 2）In obtained fibroin/hydroxyapatite powder be added to step 1）Stirred in the sericin solution of preparation
It is well mixed, obtained silk gum/(fibroin/hydroxyapatite) suspension, wherein sericin and fibroin/hydroxyapatite powder
Mass ratio be 1:0.5-1；
4）By step 3）Obtained silk gum/(fibroin/hydroxyapatite) suspension, is slowly added into chitosan solution, stirs
Mix uniform, the temperature of adjustment system is 1-10 DEG C, then step 1 is added dropwise）The phosphoglycerol sodium solution stirring 30min of preparation,
Obtain mixed solution；Wherein silk gum/(fibroin/hydroxyapatite) suspension, chitosan solution, the volume of phosphoglycerol sodium solution
Than for（0.67-1）:（5-6）:1；
5）By the 4th）Walk obtained mixed solution to pour into mould, mould is put into 37-38 DEG C of water bath with thermostatic control, makes mixed solution
Complete molten-gel conversion, precooling 2-5h at a temperature of gel rubber material is firstly placed on into -10--20 DEG C, defrosting 1-3h, Ran Houzhuan
6-10h is freezed into -70--80 DEG C of refrigerator, freeze drier is then placed in and dries more than 24h, vacuum degree control is in 10-50
Kpa, produces bone tissue engineering stent material.
2. bone tissue engineering stent material preparation method according to claim 1, it is characterised in that：Step 1）Described in silk
The preparation method of fibroin powder is：It is using the molten enzymolysis process of salt, useless light defective cocoon shell or silk is organic molten through ethers, alcohols successively
Agent immersion to remove waxy substance therein, carbohydrate and ash impurities, then through water boil degumming salt it is molten after freeze-drying i.e.
Obtain micron order silk fibroin powder.
3. bone tissue engineering stent material preparation method according to claim 1, it is characterised in that step 1）Middle silk gum egg
White powder end preparation method be：Cocoon shell is soaked into 24-48h respectively successively through ethers, alcohols, with distilled water clean, dry, with except
Remove wax and partial organic substances and impurity；Cocoon shell distilled water is boiled at least 6h again, sericin is dissolved in distilled water, taken out
Filter obtains sericin solution, and the concentration of sericin solution, freeze-drying then are produced into sericin powder.
4. the preparation method of bone tissue engineering stent material according to claim 1, it is characterised in that step 1）In it is dilute
Acid is watery hydrochloric acid or spirit of vinegar, and concentration is 0.05-0.2mol/L.
5. a kind of bone tissue engineering stent material, it is characterised in that using any described preparation method system of Claims 1 to 4
Standby to obtain, its aperture is 10~200 μm.
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| CN105597155A (en) * | 2015-12-30 | 2016-05-25 | 湖北赛罗生物材料有限责任公司 | Method for preparing hydroxyapatite-natural macromolecular nano compound with one-pot method |
| CN107412860A (en) * | 2017-08-09 | 2017-12-01 | 张家港市沐和新材料技术开发有限公司 | A kind of preparation method based on hydroxyapatite compound support frame material |
| CN111875691B (en) * | 2020-08-06 | 2022-05-17 | 苏州大学 | Preparation method of silk fibroin and sericin composite solution |
| CN111870740B (en) * | 2020-08-06 | 2021-12-24 | 苏州大学 | Composite protein film and preparation method thereof |
| CN111671980B (en) * | 2020-08-06 | 2021-12-24 | 苏州大学 | Bionic composite bone scaffold and preparation method thereof |
| CN115845148A (en) * | 2022-12-07 | 2023-03-28 | 中国医学科学院北京协和医院 | Thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102302804A (en) * | 2011-09-05 | 2012-01-04 | 济宁学院 | Hydroxyapatite-based biological composite scaffold and tissue engineered bone |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102302804A (en) * | 2011-09-05 | 2012-01-04 | 济宁学院 | Hydroxyapatite-based biological composite scaffold and tissue engineered bone |
| CN103948963A (en) * | 2014-04-25 | 2014-07-30 | 重庆理工大学 | Tissue engineering bracket applicable to human body organ construction and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 丝素/壳聚糖/纳米羟基磷灰石构建的骨组织工程支架;叶鹏等;《中国组织工程研究》;20130716;第17卷(第29期);5269-5274 * |
| 丝胶蛋白应用于组织工程支架材料的研究进展;陈忠敏等;《丝绸》;20150430;第52卷(第4期);25-30 * |
| 仿生合成丝素蛋白/羟基磷灰石类骨质复合生物材料的研究;牛林;《中国博士学位论文全文数据库 医药卫生科技辑》;20080415(第04期);第2-4、14-18、60-62页 * |
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