CN107715172A - A kind of preparation method of bone bionic composite material - Google Patents
A kind of preparation method of bone bionic composite material Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
Abstract
The present invention relates to a kind of preparation method of bone bionic composite material, belong to technical field of biological materials.The present invention uses silk to extract fibroin albumen for raw material,And the timbering material surface nucleation and oriented growth prepared with its induction hydroxyapatite crystal in native hydroxyl apatite powder,Structure simulation natural bone laminar film,It may advantageously facilitate sticking and sprawling for mesenchymal stem cells MSCs,Bone growth factor is adhered to by Dopamine hydrochloride again,The composite of 3-dimensional multi-layered orthohormbic structure is combined into simulation natural bone laminar film again,Remarkably promote the adhesion of cell,Propagation,Can also inducing bone mesenchymal stem cell to osteoblast differentiation,Manufactured bone bionic composite material high mechanical strength,Modulus of elasticity is high with skeleton matching degree,Cytoactive is high,With the good biocompatibility of human body,There is good bone-inducting active simultaneously,Reparation and the reconstruction of bone tissue can be completed faster,And biological degradability is preferable,It is worth of widely use.
Description
Technical field
The present invention relates to a kind of preparation method of bone bionic composite material, belong to technical field of biological materials.
Background technology
Bionic structure biomaterial is otherwise known as bionical biomedical material, be using natural or artificial material as skeleton, with
For the purpose of medical treatment, for being contacted with tissue of biological cells to form the material with same or similar biological function.Bionical biology
Medical material is that one kind is used for direct supplemental diagnostics, treatment, reparation or replaces live tissue, organ, promotes the new of its function
Type hitech materials.The development trend of material is Composite, intellectuality, activeization, environmental recovery bonds.And bionic structure material has simultaneously
There is the feature of this several respect.The development of bionic structure materialogy and achievement will have influence on each corner of society, be not only human body
The displacement of organ and the artificial improvement of organism system bring change, and the preparation and application generation that make material is revolutionary
Impetus.
At present, biomedical material species is various, and by sources division can be divided into natural biologic material and bioartificial materials
Two major classes.Natural biologic material is typically derived from organism itself organ transplant and allosome organ transplant, including structural proteins, life
Thing soft tissue, biological composite fibre and biomineralization thing etc..Bionic structure biomaterial, be otherwise known as artificial bionic biology material
Material.Its species is various, have the advantages that selectable range it is extensive, it is reproducible, large-scale production and application can be realized.Knot
Structure biomimetic biomaterial includes some man-made organs, body implanting material, artificial skelecton, tissue engineering material etc..In organism
The tissues such as muscle, bone, tooth with the presence of nanoscale material so that nanostructured biomimetic biomaterial is in Bone Defect Repari
Occupy critical role in material.
Existing bone tissue engineer cell epimatrix material respectively has advantage and disadvantage.Natural derived material is as bone tissue engineer
Timbering material, its advantage are good good biocompatibilities, can form the loose structure similar with people's bone, and its catabolite
It is easy to be absorbed without generation inflammatory reaction.But its shortcoming is to be difficult to shaping, poor mechanical property, process complexity and labor
Momentum is big, after processing in polymer matrix residual particles be difficult to remove, degradation rate and skeletonization speed is uncoordinated and different times
Product quality disunity of different batches etc..Its theoretical research and application are clinically had a strong impact on.
At present, bionic structure skeleton material mainly has bioceramic material, metal coating layer material etc..Wherein biology pottery
Ceramic material has the advantages that good biocompatibility and bioactivity, stable in physicochemical property, human body is had no toxic side effect, but makes pottery
Particle possessed by porcelain powder is smaller, easy accumulation, it is easily hardened after sintering the shortcomings of be difficult to solve so far.And metal coating layer material has
Have the advantages that hardness is big, wear-resistant, outward appearance is beautiful, but metal material is perishable under acid or alkali environment, service life is short and golden
Category ion into human body toxic side effect etc. is caused to human body the shortcomings that.Composite construction biomimetic biomaterial be by two kinds or two kinds with
On Material cladding form, combine the advantage in different materials structure and performance, overcome structure existing for homogenous material and property
The defects of can, and the performance of composite various aspects can be controlled by regulating and controlling composition and experiment condition, it is further full
Sufficient people to the different demands of material property, but existing composite construction biomimetic biomaterial there is also mechanical strength it is too high,
Modulus of elasticity is not high with skeleton matching degree, the shortcomings of cell compatibility and cytoactive difference, also needs continual exploitation to study.
The content of the invention
The technical problems to be solved by the invention：For existing composite construction biomimetic biomaterial, there is also machinery is strong
Spend high, modulus of elasticity and skeleton matching degree be not high, the problem of the shortcomings of cell compatibility and cytoactive difference, there is provided
A kind of preparation method of bone bionic composite material.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of preparation method of bone bionic composite material, it is characterised in that specifically preparation process is：
（1）By the clean rear sodium carbonate liquor boiling 3~5 times of silk washing, boiled silk is obtained；
（2）Boiled silk is added in calcium chloride solution, adds after ethanol is well mixed to be fitted into bag filter and dialyses 6~8 days,
Every 4~8h changes deionized water, and dialysis is filtered and is freeze-dried after terminating, and obtains fibroin albumen；
（3）1~2h will be calcined after fresh ox bone drying and crushing at 600~700 DEG C, obtain calcined product；
（4）Take calcined product, CPP, carboxymethyl cellulose to be fitted into 30~40min of ball milling in ball mill, cross 200 mesh
Sieve, obtains composite natral hydroxyapatite powder；
（5）It is fitted into after composite natral hydroxyapatite powder is mixed with deionized water in mould, and is suppressed with 3~5MPa pressure
Shaping, dry timbering material；
（6）Take tertiary sodium phosphate, calcium chloride, fibroin albumen, deionized water to be configured to mineralized liquid, and soak 10~20h of timbering material,
Obtain remineralization timbering material；
（7）Dopamine hydrochloride, bone growth factor are taken, adds in tris-HCI buffer and is well mixed, and soak
Remineralization timbering material, then culture growth 7~10 days at 36~37 DEG C are steeped, timbering material is taken out and sterilized after drying, obtains bone
Bionic composite material.
Step（1）The mass fraction of the sodium carbonate liquor is 0.1~0.5%, and dosage is 10~100 times of silk quality.
Step（2）The mass fraction of the calcium chloride solution is 10%, and dosage is 5~20 times of boiled silk, the ethanol
Dosage is 1~4 times of boiled silk.
Step（4）The calcined product, CPP, the parts by weight of carboxymethyl cellulose are 200~300 parts of calcinings
Product, 100~200 parts of CPPs, 5~10 parts of carboxymethyl celluloses.
Step（5）The composite natral hydroxyapatite powder and the mass ratio of deionized water are 4：1.
Step（6）The mineralized liquid each component parts by weight are 10~15 parts of tertiary sodium phosphates, 10~15 parts of calcium chloride, 3~5 parts
Fibroin albumen, 300~500 parts of deionized waters.
Step（7）The Dopamine hydrochloride, bone growth factor, the parts by weight of tris-HCI buffer
For 3~5 parts of Dopamine hydrochlorides, 1~2 part of bone growth factor, 300~500 parts of pH are 7.5~8.5 trishydroxymethylaminomethanes-salt
Acid buffer.
Compared with other method, advantageous effects are the present invention：
The present invention uses silk to extract fibroin albumen for raw material, and induces hydroxyapatite crystal in native hydroxyl apatite with it
Timbering material surface nucleation and oriented growth prepared by powder, structure simulation natural bone laminar film, may advantageously facilitate between marrow
Mesenchymal stem cells are sticked and sprawled, then adhere to bone growth factor by Dopamine hydrochloride, then with simulating natural bone laminar film
The composite of 3-dimensional multi-layered orthohormbic structure is combined into, remarkably promotes adhesion, the propagation of cell, additionally it is possible to inducing bone mesenchymal
The differentiation of stem cell to osteoblast, manufactured bone bionic composite material high mechanical strength, modulus of elasticity match with skeleton
Degree is high, and cytoactive is high, the good biocompatibility with human body, while has good bone-inducting active, can complete faster
The reparation of bone tissue and reconstruction, and biological degradability is preferable, is worth of widely use.
Embodiment
Silk is washed with deionized 2~3 times and is fitted into baking oven, 1~2h is dried at 55~60 DEG C, takes 20~30g
Silk, adding 2~3L mass fractions is in 0.5% sodium carbonate liquor, and is heated to 95~100 DEG C of 30~40min of boiling, filters
Filter residue and filter residue is washed with deionized 2~3 times, be placed in after repeating boiling 3~5 times in drying box, at 70~80 DEG C
2~3h is dried, obtains boiled silk, takes 5~10g boiled silks, adding 50~100g mass fractions is in 10% calcium chloride solution,
20~30min is stirred with 300~400r/min, 10~20g ethanol is added, loads bag filter after continuing 20~30min of stirring
Middle dialysis 6~8 days, every 4~8h change deionized water, and dialysis filters to get filtrate after terminating, filtrate is placed in freeze drying box
10~20h is freeze-dried, fibroin albumen is obtained, takes the fresh ox bones of 1~2kg, be placed in drying box, is dried extremely at 105~110 DEG C
Constant weight, then dried ox bone is fitted into pulverizer and crushed, 100 mesh sieves are crossed, the bovine bone powder after sieving is placed in Muffle furnace,
1~2h is calcined at 600~700 DEG C, after being cooled to room temperature, calcined product is obtained, takes 200~300g calcined products, 100~200g
CPP, 5~10g carboxymethyl celluloses are transferred in ball mill, with 200~300r/min, 30~40min of ball milling, mistake
200 mesh sieves, composite natral hydroxyapatite powder is obtained, take 80~100g composite natrals hydroxyapatite powder to be gone with 20~25mL
It is fitted into after ionized water mixing in mould, it is and compressing with 3~5MPa pressure, then mould is placed in drying box, 105~
Dried at 110 DEG C to constant weight, be stripped after being cooled to room temperature, obtain timbering material, take 10~15g tertiary sodium phosphates, 10~15g chlorinations
Calcium, 3~5g fibroin albumens, add in 300~500mL deionized waters, 20~30min is stirred with 300~400r/min, added
Timbering material, 10~20h is soaked at 50~60 DEG C, take out timbering material, obtain remineralization timbering material, take 3~5g hydrochloric acid more
Bar amine, 1~2g bone growth factors, it is 7.5~8.5 to add pH in 300~500g tris-HCI buffers,
20~30min is stirred with 300~400r/min, and soaks remineralization timbering material, then is transferred in climatic chamber, 36~37
After culture grows 7~10 days at DEG C, timbering material is taken out and sterilized after drying, obtains bone bionic composite material.
Example 1
Silk is washed with deionized 2 times and is fitted into baking oven, 1h is dried at 55 DEG C, take 20g silks, add 2L mass fractions
For in 0.5% sodium carbonate liquor, and 95 DEG C of 30~40min of boiling are heated to, filter filter residue and filter residue 2 to be washed with deionized
It is secondary, it is placed in after repeating boiling 3 times in drying box, 2h is dried at 70 DEG C, obtains boiled silk, take 5g boiled silks, adds 50g
Mass fraction is in 10% calcium chloride solution, stirs 20min with 300r/min, adds 10g ethanol, continues to fill after stirring 20min
Enter in bag filter and dialyse 6 days, deionized water is changed per 4h, dialysis is filtered to get filtrate after terminating, and filtrate is placed in into freeze drying box
Middle freeze-drying 10h, obtains fibroin albumen, takes the fresh ox bones of 1kg, is placed in drying box, is dried at 105 DEG C to constant weight, then will
Dried ox bone, which is fitted into pulverizer, to be crushed, and is crossed 100 mesh sieves, the bovine bone powder after sieving is placed in Muffle furnace, at 600 DEG C
1h is calcined, after being cooled to room temperature, calcined product is obtained, takes 200g calcined products, 100g CPPs, 5g carboxymethyl celluloses
It is transferred in ball mill, with 200r/min ball milling 30min, crosses 200 mesh sieves, obtain composite natral hydroxyapatite powder, take 80g compound
Native hydroxyl apatite powder is fitted into mould after being mixed with 20mL deionized waters, and compressing with 3MPa pressure, then by mould
Tool is placed in drying box, is dried at 105 DEG C to constant weight, is stripped after being cooled to room temperature, obtains timbering material, take 10g tertiary sodium phosphates,
10g calcium chloride, 3g fibroin albumens, add in 300mL deionized waters, 20min stirred with 300r/min, adds timbering material,
10h is soaked at 50 DEG C, timbering material is taken out, obtains remineralization timbering material, take 3g Dopamine hydrochlorides, 1g bone growth factors, add
It is 7.5 to enter pH in 300g tris-HCI buffers, stirs 20min with 300r/min, and soak remineralization branch
Frame material, then be transferred in climatic chamber, after culture grows 7 days at 36 DEG C, timbering material is taken out and sterilized after drying, obtains bone
Bionic composite material.
Example 2
Silk is washed with deionized 2 times and is fitted into baking oven, 1h is dried at 58 DEG C, take 25g silks, add 2L mass fractions
For in 0.5% sodium carbonate liquor, and 98 DEG C of boiling 35min are heated to, filter filter residue and filter residue to be washed with deionized 2 times, then
It is placed in after repeating boiling 4 times in drying box, 2h is dried at 75 DEG C, obtains boiled silk, take 8g boiled silks, adds 80g mass
Fraction is in 10% calcium chloride solution, stirs 25min with 350r/min, adds 15g ethanol, continues to load thoroughly after stirring 25min
Dialysed 7 days in analysis bag, deionized water is changed per 6h, dialysis filters to get filtrate after terminating, filtrate is placed in cold in freeze drying box
Dry 15h is freezed, fibroin albumen is obtained, takes the fresh ox bones of 1kg, be placed in drying box, is dried at 108 DEG C to constant weight, then by drying
Ox bone afterwards, which is fitted into pulverizer, to be crushed, and crosses 100 mesh sieves, the bovine bone powder after sieving is placed in Muffle furnace, calcined at 650 DEG C
1h, after being cooled to room temperature, calcined product is obtained, take 250g calcined products, 150g CPPs, 8g carboxymethyl celluloses are transferred to
In ball mill, with 250r/min ball milling 35min, 200 mesh sieves are crossed, composite natral hydroxyapatite powder is obtained, takes 90g composite natrals
Hydroxyapatite powder is fitted into mould after being mixed with 22mL deionized waters, and compressing with 4MPa pressure, then mould is put
In drying box, dried at 108 DEG C to constant weight, be stripped after being cooled to room temperature, obtain timbering material, take 12g tertiary sodium phosphates, 12g
Calcium chloride, 4g fibroin albumens, add in 400mL deionized waters, 25min is stirred with 350r/min, timbering material is added, 55
15h is soaked at DEG C, timbering material is taken out, obtains remineralization timbering material, 4g Dopamine hydrochlorides is taken, 1g bone growth factors, adds
PH is 8.0 in 400g tris-HCI buffers, stirs 25min with 350r/min, and soak remineralization support
Material, then be transferred in climatic chamber, after culture grows 9 days at 36 DEG C, timbering material is taken out and sterilized after drying, bone is obtained and imitates
Raw composite.
Example 3
Silk is washed with deionized 3 times and is fitted into baking oven, 2h is dried at 60 DEG C, take 30g silks, add 3L mass fractions
For in 0.5% sodium carbonate liquor, and 100 DEG C of boiling 40min are heated to, filter filter residue and filter residue to be washed with deionized 3 times,
It is placed in after repeating boiling 5 times in drying box, 3h is dried at 80 DEG C, obtains boiled silk, take 10g boiled silks, adds 100g
Mass fraction is in 10% calcium chloride solution, stirs 30min with 400r/min, adds 20g ethanol, continues to fill after stirring 30min
Enter in bag filter and dialyse 8 days, deionized water is changed per 8h, dialysis is filtered to get filtrate after terminating, and filtrate is placed in into freeze drying box
Middle freeze-drying 20h, obtains fibroin albumen, takes the fresh ox bones of 2kg, is placed in drying box, is dried at 110 DEG C to constant weight, then will
Dried ox bone, which is fitted into pulverizer, to be crushed, and is crossed 100 mesh sieves, the bovine bone powder after sieving is placed in Muffle furnace, at 700 DEG C
2h is calcined, after being cooled to room temperature, calcined product is obtained, takes 300g calcined products, 200g CPPs, 10g carboxymethyl celluloses
Element is transferred in ball mill, with 300r/min ball milling 40min, is crossed 200 mesh sieves, is obtained composite natral hydroxyapatite powder, take 100g
Composite natral hydroxyapatite powder is fitted into mould after being mixed with 25mL deionized waters, and compressing with 5MPa pressure, then
Mould is placed in drying box, is dried at 110 DEG C to constant weight, is stripped after being cooled to room temperature, obtains timbering material, take 15g phosphoric acid
Trisodium, 15g calcium chloride, 5g fibroin albumens, add in 500mL deionized waters, 30min is stirred with 400r/min, adds support
Material, soaks 20h at 60 DEG C, takes out timbering material, obtains remineralization timbering material, takes 5g Dopamine hydrochlorides, 2g bone uptakes because
Son, it is 8.5 to add pH in 500g tris-HCI buffers, stirs 30min with 400r/min, and soak again
Mineralising timbering material, then be transferred in climatic chamber, after culture grows 10 days at 37 DEG C, timbering material is taken out and gone out after drying
Bacterium, obtain bone bionic composite material.
Reference examples：The biomimetic material of Suzhou company production.
The biomimetic material of example and reference examples is detected, specific detection is as follows：
Carry out the toughness of research material from S-4800 type ESEMs.
The compressive property of material is probed into from AFM.
By calculating to obtain single nanofiber Deflection Modulus of Elasticity k=13.5-25.0GP, this and skeleton bending resistance are elastic
Modulus k=17-18.9GP is identical.Test result shows, in the case of applying active force in outside, biomimetic type nanometer bone
Material has good mechanical strength and high-quality toughness.This is to accord with completely with the intensity required for natural bone material and toughness
Close.
The pressure-proof elasticity modulus of biomimetic type nanometer bone material between nanofiber, its pressure-proof elasticity modulus with it is natural
The pressure-proof elasticity modulus of bone are similar, and exceed the compression strength of native human's bone, in the case where applying external force, receiving
The effect of external force is disperseed, buffered to the nano-pillar of rice fiber surface oriented growth so that the resistance to compression of biomimetic type nanometer bone material
Intensity increases considerably.When external force eliminates, the toughness as the nanofiber of main body plays a role so that biomimetic type nano bone
Bone material restores to the original state.This structure make it that high intensity and high tenacity are mutually compound, at the same draw both the advantages of, make up both sides'
Shortcoming.Realize the bionical of structure and function.
In biocompatibility test, after cell culture one day, start to be dispersed in the surface of this nanometer of bone, with cell
The increase of incubation time, the quantity of cell are continuously increased, and surface and the internal cavities of full material are finally completely covered.Thus table
Bright, biomimetic type nanometer bone material has excellent biocompatibility.Its unique mesh-structured and biocompatibility is good
With reference to can promote the sticking of Gegenbaur's cell, breed and differentiation.
Claims (7)
1. a kind of preparation method of bone bionic composite material, it is characterised in that specifically preparation process is：
（1）By the clean rear sodium carbonate liquor boiling 3~5 times of silk washing, boiled silk is obtained；
（2）Boiled silk is added in calcium chloride solution, adds after ethanol is well mixed to be fitted into bag filter and dialyses 6~8 days,
Every 4~8h changes deionized water, and dialysis is filtered and is freeze-dried after terminating, and obtains fibroin albumen；
（3）1~2h will be calcined after fresh ox bone drying and crushing at 600~700 DEG C, obtain calcined product；
（4）Take calcined product, CPP, carboxymethyl cellulose to be fitted into 30~40min of ball milling in ball mill, cross 200 mesh
Sieve, obtains composite natral hydroxyapatite powder；
（5）It is fitted into after composite natral hydroxyapatite powder is mixed with deionized water in mould, and is suppressed with 3~5MPa pressure
Shaping, dry timbering material；
（6）Take tertiary sodium phosphate, calcium chloride, fibroin albumen, deionized water to be configured to mineralized liquid, and soak 10~20h of timbering material,
Obtain remineralization timbering material；
（7）Dopamine hydrochloride, bone growth factor are taken, adds in tris-HCI buffer and is well mixed, and soak
Remineralization timbering material, then culture growth 7~10 days at 36~37 DEG C are steeped, timbering material is taken out and sterilized after drying, obtains bone
Bionic composite material.
A kind of 2. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（1）The carbon
The mass fraction of acid sodium solution is 0.1~0.5%, and dosage is 10~100 times of silk quality.
A kind of 3. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（2）The chlorine
The mass fraction for changing calcium solution is 10%, and dosage is 5~20 times of boiled silk, and the ethanol consumption is the 1~4 of boiled silk
Times.
A kind of 4. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（4）It is described to forge
Burn product, CPP, the parts by weight of carboxymethyl cellulose are 200~300 parts of calcined products, 100~200 parts of caseins
Phosphoeptide, 5~10 parts of carboxymethyl celluloses.
A kind of 5. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（5）It is described multiple
The mass ratio for closing native hydroxyl apatite powder and deionized water is 4：1.
A kind of 6. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（6）The ore deposit
It is 10~15 parts of tertiary sodium phosphates to change liquid each component parts by weight, and 10~15 parts of calcium chloride, 3~5 parts of fibroin albumens, 300~500 parts are gone
Ionized water.
A kind of 7. preparation method of bone bionic composite material as claimed in claim 1, it is characterised in that step（7）The salt
Sour dopamine, bone growth factor, the parts by weight of tris-HCI buffer are 3~5 parts of Dopamine hydrochlorides, 1~
2 parts of bone growth factors, 300~500 parts of pH are 7.5~8.5 tris-HCI buffers.
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| CN111097071A (en) * | 2019-11-28 | 2020-05-05 | 上海摩漾生物科技有限公司 | Porous material, casein calcium phosphorus microsphere and preparation method and application thereof |
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